Compositions and methods relating to non-human animals modified to promote production of selected gametes

ABSTRACT

Methods and compositions for producing selected non-human mammalian germ cells and gametes and for making non-human animals using the produced germ cells and gametes are provided by the present invention. Methods of generating a non-human embryo and/or animal derived from donor stem cells, methods of generating chimeric non-human animals having substantially all gametes and/or germ cells derived from the donor stem cells, methods of producing a non-human host embryo lacking functional endogenous germ cells and non-human host embryos incapable of developing endogenous gametes of the present invention are described herein.

REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional PatentApplication Ser. No. 61/427,337, filed Dec. 27, 2010, the entire contentof which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. R21RR031289 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions forproducing selected non-human mammalian germ cells and gametes and formaking non-human animals using the produced germ cells and gametes.According to specific aspects the present invention relates to methodsof generating a non-human embryo and/or animal derived from donor stemcells, methods of generating chimeric non-human animals havingsubstantially all gametes and/or germ cells derived from the donor stemcells, methods of producing a non-human host embryo lacking functionalendogenous germ cells and a non-human host embryo incapable ofdeveloping endogenous gametes.

BACKGROUND OF THE INVENTION

Prior to the present invention, the efficiency of stem cells to populatethe germline and to produce non-human animals has been a majorchallenge, and there has been a continuing need for methods andcompositions for the production of non-human animals from stem cells,including production of genetically modified non-human animals fromgenetically modified stem cells. This invention provides methods andcompositions to produce non-human host embryos having a receptive nichefor the development of donor stem cells, including genetically modifieddonor stem cells, into germ cells and gametes.

SUMMARY OF THE INVENTION

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) producing a preimplantation non-human hostembryo incapable of developing endogenous gametes; b) introducing donorstem cells into the preimplantation non-human host embryo; c) gestatingthe non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

The terms “endogenous gametes” and “endogenous germ cells” as usedherein refer to gametes and germ cells “originating or produced fromwithin” a host embryo and exclude gametes and germ cells in the hostembryo which are derived from donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expressing acytotoxic protein in germ cells of the non-human host embryo to ablateendogenous germ cells; b) introducing donor stem cells into thepreimplantation non-human host embryo; c) gestating the non-human hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric non-human animal having substantially allgametes and/or germ cells derived from the donor stem cells; and d)making a non-human embryo or animal using the gametes and/or germ cellsderived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expressing aninhibitory RNA in germ cells of the non-human host embryo to ablateendogenous germ cells; b) introducing donor stem cells into thepreimplantation non-human host embryo; c) gestating the non-human hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric non-human animal having substantially allgametes and/or germ cells derived from the donor stem cells; and d)making a non-human embryo or animal using the gametes and/or germ cellsderived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expressing arecombinase to excise two inverted recombinase recognition sites placedin a chromosome of a preimplantation non-human host embryo to ablate theendogenous germ cells; b) introducing donor stem cells into thepreimplantation non-human host embryo; c) gestating the non-human hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric non-human animal having substantially allgametes and/or germ cells derived from the donor stem cells; and d)making a non-human embryo or animal using the gametes and/or germ cellsderived from the donor stem cells.

A preimplantation non-human host embryo produced according to methodsdescribed herein is incapable of developing endogenous gametes due tohuman intervention using methods of the present invention, such asexpressing a cytotoxic protein in germ cells of the non-human hostembryo to ablate endogenous germ cells; expressing an inhibitory RNA ingerm cells of the non-human host embryo to ablate endogenous germ cells;and placing two inverted recombinase recognition sites in a chromosomeof a preimplantation non-human host embryo and expressing a recombinaseto ablate the endogenous germ cells.

As described herein, donor stem cells are introduced into thepreimplantation non-human host embryo. As described herein, the donorstem cells may be introduced before ablation of the endogenous germcells such that it will be appreciated that the endogenous germ cells inthe preimplantation non-human host embryo can be referred to as“lacking, or destined to lack endogenous germ cells.”

A preimplantation non-human host embryo can be a 2-cell stage embryo, a4-cell stage embryo, a 8-cell stage embryo, a 16-cell stage embryo, a32-cell stage embryo, a 64-cell stage embryo, a morula or a blastocyst.The inner cell mass of preimplantation non-human host embryos is notablated according to methods of the present invention.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding a cytotoxic proteinoperably linked to a developmentally regulated promoter active in germcells of the embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice, to ablatethe endogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a non-human embryo or animal using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding an inhibitory RNAoperably linked to a developmentally regulated promoter active in germcells of the embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice, to ablatethe endogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a non-human embryo or animal using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes at least two transgenes,one transgene including a nucleic acid sequence encoding a recombinaselinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice, to ablatethe endogenous germ cells of the embryo and the second transgeneincluding a nucleic acid sequence encoding a cytotoxic protein or aninhibitory RNA operably linked to recombinase recognition sites; b)introducing donor stem cells into the preimplantation non-human hostembryo; c) gestating the non-human host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericnon-human animal having substantially all gametes and/or germ cellsderived from the donor stem cells; and d) making a non-human embryo oranimal using the gametes and/or germ cells derived from the donor stemcells.

Methods of generating a rodent embryo and/or rodent derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation rodent hostembryo incapable of developing endogenous gametes wherein thepreimplantation rodent host embryo includes a transgene, the transgeneincluding a nucleic acid sequence encoding a cytotoxic protein operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryo,to ablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation rodent host embryo; c) gestating therodent host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric rodent having substantiallyall gametes and/or germ cells derived from the donor stem cells; and d)making a rodent embryo and/or rodent using the gametes and/or germ cellsderived from the donor stem cells.

Methods of generating a rodent embryo and/or rodent derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation rodent hostembryo incapable of developing endogenous gametes wherein thepreimplantation rodent host embryo includes a transgene, the transgeneincluding a nucleic acid sequence encoding an inhibitory RNA operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryo,to ablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation rodent host embryo; c) gestating therodent host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric rodent having substantiallyall gametes and/or germ cells derived from the donor stem cells; and d)making a rodent embryo and/or rodent using the gametes and/or germ cellsderived from the donor stem cells.

Methods of generating a rodent embryo and/or rodent derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation rodent hostembryo incapable of developing endogenous gametes wherein thepreimplantation rodent host embryo includes a recombinase operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryoto excise two inverted recombinase recognition sites placed in achromosome of a preimplantation non-human host embryo to ablate theendogenous germ cells, to ablate the endogenous germ cells of theembryo; b) introducing donor stem cells into the preimplantation rodenthost embryo; c) gestating the rodent host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericrodent having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a rodent embryo and/or rodent usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a mouse embryo and/or mouse derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation mouse hostembryo incapable of developing endogenous gametes wherein thepreimplantation mouse host embryo includes a transgene, the transgeneincluding a nucleic acid sequence encoding a cytotoxic protein operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during a portion of embryonic day 6 to embryonic day 14 of amouse embryo, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation mouse host embryo;c) gestating the mouse host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric mouse havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a mouse embryo and/or mouse using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a mouse embryo and/or mouse derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation mouse hostembryo incapable of developing endogenous gametes wherein thepreimplantation mouse host embryo includes a transgene, the transgeneincluding a nucleic acid sequence encoding an inhibitory RNA operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during a portion of embryonic day 6 to embryonic day 14 of amouse embryo, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation mouse host embryo;c) gestating the mouse host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric mouse havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a mouse embryo and/or mouse using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a mouse embryo and/or mouse derived from donorstem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation mouse hostembryo incapable of developing endogenous gametes wherein thepreimplantation mouse host embryo includes at least two transgenes, onetransgene including a nucleic acid sequence encoding a recombinaseoperably linked to a developmentally regulated promoter active in germcells of the embryo during a portion of embryonic day 6 to embryonic day14 of a mouse embryo, and the second transgene including a nucleic acidsequence encoding a cytotoxic protein or an inhibitory RNA operablylinked to recombinase recognition sites to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation mouse host embryo; c) gestating the mouse host embryo ofb) under conditions suitable for development of the embryo, therebygenerating a chimeric mouse having substantially all gametes and/or germcells derived from the donor stem cells; and d) making a mouse embryoand/or mouse using the gametes and/or germ cells derived from the donorstem cells.

Methods of generating a rat embryo and/or rat derived from donor stemcells, are provided according to embodiments of the present inventionwhich include: a) generating a preimplantation rat host embryo incapableof developing endogenous gametes wherein the preimplantation rat hostembryo includes a transgene, the transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring a portion of embryonic day 7 to embryonic day 16 of a rat embryo,to ablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation rat host embryo; c) gestating therat host embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric rat having substantially allgametes and/or germ cells derived from the donor stem cells; and d)making a rat embryo and/or mouse using the gametes and/or germ cellsderived from the donor stem cells.

Methods of generating a rat embryo and/or rat derived from donor stemcells, are provided according to embodiments of the present inventionwhich include: a) generating a preimplantation rat host embryo incapableof developing endogenous gametes wherein the preimplantation rat hostembryo includes a transgene, the transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter active in germ cells of the embryo during a portionof embryonic day 7 to embryonic day 16 of a rat embryo, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation rat host embryo; c) gestating the rat hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric rat having substantially all gametesand/or germ cells derived from the donor stem cells; and d) making a ratembryo and/or mouse using the gametes and/or germ cells derived from thedonor stem cells.

Methods of generating a rat embryo and/or rat derived from donor stemcells, are provided according to embodiments of the present inventionwhich include: a) generating a preimplantation rat host embryo incapableof developing endogenous gametes wherein the preimplantation rat hostembryo includes at least two transgenes, one transgene including anucleic acid sequence encoding a recombinase operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring a portion of embryonic day 7 to embryonic day 16 of a rat embryo,and the second transgene including a nucleic acid sequence encoding acytotoxic protein or an inhibitory RNA operably linked recombinaserecognition sites to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation rat host embryo;c) gestating the rat host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric rat havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a rat embryo and/or mouse using the gametes and/orgerm cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expression of atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter or a ubiquitous promoter, the transgene further including atleast one cytotoxic protein inhibitory sequence operably linked to atleast two recombinase recognition sites; wherein the non-human hostembryo further includes a second transgene including a nucleic acidsequence encoding a recombinase, the nucleic acid sequence encoding therecombinase operably linked to a promoter selected from the groupconsisting of: a developmentally regulated promoter and a ubiquitouspromoter, wherein at least either the first or second transgene isoperably linked to a developmentally regulated promoter; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expression of atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to recombinase recognition sites and to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further including at least one inhibitory RNA inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further includes a second transgeneincluding a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a promoterselected from the group consisting of: a developmentally regulatedpromoter and a ubiquitous promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter; b) introducing donor stem cells into the preimplantationnon-human host embryo; c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells; and d) making anon-human embryo or animal using the gametes and/or germ cells derivedfrom the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding a cytotoxic proteinoperably linked to an inducible promoter, an inhibitory RNA operablylinked to an inducible promoter or a recombinase operably linked to aninducible promoter, wherein the recombinanse is active to excise twoinverted recombinase recognition sites placed in a chromosome of apreimplantation non-human host embryo, to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation non-human host embryo; c) gestating the non-human hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric non-human animal having 80% or moregametes and/or germ cells of the chimeric non-human animal are derivedfrom the donor stem cells; and d) making a non-human embryo or animalusing the gametes and/or germ cells derived from the donor stem cells.

Chimeric non-human animals generated according to methods describedherein can have substantially all gametes and/or germ cells of thechimeric non-human animal derived from the donor stem cells such as 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more gametes and/or germ cells of thechimeric non-human animal which are derived from the donor stem cells.

Donor stem cells introduced into a preimplantation non-human host embryoincapable of developing endogenous gametes are embryonic stem cells,epiblast stem cells, embryonic germ cells, induced pluripotent stemcells, genetically modified embryonic stem cells, genetically modifiedepiblast stem cells, genetically modified embryonic germ cells,genetically modified induced pluripotent stem cells or a combination ofany two or more of these.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expression of afirst transgene, the first transgene including a nucleic acid sequenceencoding a cytotoxic protein operably linked to a ubiquitous promoter ora developmentally regulated promoter selected from the group consistingof: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2 promoter,Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiar promoter, thetransgene further including at least one cytotoxic protein inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further includes a second transgeneincluding a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a ubiquitouspromoter or a developmentally regulated promoter selected from the groupconsisting of: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2promoter, Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiarpromoter, wherein at least either the first or second transgene isoperably linked to a developmentally regulated promoter; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expression of afirst transgene, the first transgene including a nucleic acid sequenceencoding an inhibitory RNA operably linked to a ubiquitous promoter or adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter, the transgene further including at least onecytotoxic protein inhibitory sequence operably linked to at least tworecombinase recognition sites; wherein the non-human host embryo furtherincludes a second transgene including a nucleic acid sequence encoding arecombinase, the nucleic acid sequence encoding the recombinase operablylinked to a ubiquitous promoter or a developmentally regulated promoterselected from the group consisting of: vasa promoter, c-kit promoter,Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter,Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter,Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAP promoter,wherein at least either the first or second transgene is operably linkedto a developmentally regulated promoter; b) introducing donor stem cellsinto the preimplantation non-human host embryo; c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a non-human embryo or animal using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding a cytotoxic proteinoperably linked to a developmentally regulated promoter active in germcells of the embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice selectedfrom the group consisting of: vasa promoter, Dnd1 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter, to ablate the endogenous germ cellsof the embryo; b) introducing donor stem cells into the preimplantationnon-human host embryo; c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells; and d) making anon-human embryo or animal using the gametes and/or germ cells derivedfrom the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding an inhibitory RNAoperably linked to a developmentally regulated promoter active in germcells of the embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice selectedfrom the group consisting of: vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter and TNAP promoter, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a non-human embryo or animal using the gametesand/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding a recombinaselinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice selectedfrom the group consisting of: vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter and TNAP promoter, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells; and d) making a non-human embryo or animal using the gametesand/or germ cells derived from the donor stem cells.

The preimplantation non-human host embryo is an embryo of any non-humanmammal, such as rodent, non-human primate, rabbit, dog, cat, cattle,horse, sheep, goat, endangered mammal and exotic mammal. Thepreimplantation non-human host embryo is a marmoset embryo, for example.In a further example, the rodent preimplantation host embryo is a mouseor rat embryo.

Optionally, the donor stem cells are derived from a first animal speciesand the preimplantation non-human host embryo is a different secondanimal species. In one option, the donor stem cells are rat stem cellsand the preimplantation non-human host embryo is a mouse embryo, suchthat rat gametes and/or rat germ cells are produced in a mouse hostanimal.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding Herpes simplexvirus thymidine kinase operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice and further comprising contacting endogenous germ cellsexpressing a Herpes simplex virus thymidine kinase with a thymidineanalog to ablate the endogenous germ cells of the embryo; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding Herpes simplexvirus thymidine kinase operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter, and further comprising contacting endogenous germ cellsexpressing the Herpes simplex virus thymidine kinase with a thymidineanalog to ablate the endogenous germ cells of the embryo; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding a functional mutantor truncated Herpes simplex virus thymidine kinase operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring at least a portion of a developmental stage corresponding toembryonic day 6 to embryonic day 14 in mice selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter, and further comprisingcontacting endogenous germ cells expressing the functional mutant ortruncated Herpes simplex virus thymidine kinase with a thymidine analogto ablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation non-human host embryo; c) gestatingthe non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes wherein thepreimplantation non-human host embryo includes a transgene, thetransgene including a nucleic acid sequence encoding Herpes simplexvirus thymidine kinase operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter, and further comprising contacting endogenous germ cellsexpressing the Herpes simplex virus thymidine kinase with a thymidineanalog selected from the group consisting of: ganciclovir, acyclovir andfialuridine, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation non-human hostembryo; c) gestating the non-human host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericnon-human animal having substantially all gametes and/or germ cellsderived from the donor stem cells; and d) making a non-human embryo oranimal using the gametes and/or germ cells derived from the donor stemcells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expressingdiphtheria toxin A fragment, attenuated diphtheria toxin A fragment,tox-176; or a cytotoxic homologue, fragment or variant thereof, in germcells of the non-human host embryo to ablate endogenous germ cells; b)introducing donor stem cells into the preimplantation non-human hostembryo; c) gestating the non-human host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericnon-human animal having substantially all gametes and/or germ cellsderived from the donor stem cells; and d) making a non-human embryo oranimal using the gametes and/or germ cells derived from the donor stemcells.

Methods of generating a non-human embryo and/or animal derived fromdonor stem cells, are provided according to embodiments of the presentinvention which include: a) generating a preimplantation non-human hostembryo incapable of developing endogenous gametes by expression of afirst transgene, the first transgene including a nucleic acid sequenceencoding diphtheria toxin A fragment, attenuated DTA, tox-176; or acytotoxic homologue, fragment or variant thereof, operably linked to aubiquitous promoter or a developmentally regulated promoter selectedfrom the group consisting of: vasa promoter, Dnd1 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter, the transgene further including atleast one inhibitory sequence for diphtheria toxin A fragment,attenuated DTA, tox-176; or a cytotoxic homologue, fragment or variantthereof, and operably linked to least two recombinase recognition sites;wherein the non-human host embryo further includes a second transgeneincluding a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a ubiquitouspromoter or a developmentally regulated promoter selected from the groupconsisting of: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2promoter, Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiarpromoter, wherein at least either the first or second transgene isoperably linked to a developmentally regulated promoter; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) making a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) producing apreimplantation non-human host embryo incapable of developing endogenousgametes; b) introducing donor stem cells into the preimplantationnon-human host embryo; and c) gestating the preimplantation non-humanhost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expressing a cytotoxic protein in germ cells of the non-humanhost embryo to ablate endogenous germ cells; b) introducing donor stemcells into the preimplantation non-human host embryo; and c) gestatingthe non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expressing an inhibitory RNA in germ cells of the non-humanhost embryo to ablate endogenous germ cells; b) introducing donor stemcells into the preimplantation non-human host embryo; and c) gestatingthe non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expressing a recombinase to excise two inverted recombinaserecognition sites placed in a chromosome of a preimplantation non-humanhost embryo and expressing a recombinase to ablate the endogenous germcells; b) introducing donor stem cells into the preimplantationnon-human host embryo; and c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation non-human hostembryo; and c) gestating the non-human host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric non-human animal having substantially all gametes and/or germcells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14in mice, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation non-human hostembryo; and c) gestating the non-human host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric non-human animal having substantially all gametes and/or germcells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding arecombinase linked to a developmentally regulated promoter active ingerm cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 in mice, toablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation non-human host embryo; and c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rodent host embryo incapable of developing endogenousgametes wherein the preimplantation rodent host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 of a mouse embryo, to ablate the endogenous germ cells of the embryo;b) introducing donor stem cells into the preimplantation rodent hostembryo; and c) gestating the rodent host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericrodent having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rodent host embryo incapable of developing endogenousgametes wherein the preimplantation rodent host embryo includes atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14of a mouse embryo, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation rodent hostembryo; and c) gestating the rodent host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericrodent having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rodent host embryo incapable of developing endogenousgametes wherein the preimplantation rodent host embryo includes arecombinase operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14of a mouse embryo, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation rodent hostembryo; and c) gestating the rodent host embryo of b) under conditionssuitable for development of the embryo, thereby generating a chimericrodent having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation mouse host embryo incapable of developing endogenousgametes wherein the preimplantation mouse host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during a portion ofembryonic day 6 to embryonic day 14 of a mouse embryo, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation mouse host embryo; and c) gestating the mousehost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric mouse having substantially allgametes and/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation mouse host embryo incapable of developing endogenousgametes wherein the preimplantation mouse host embryo includes atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during a portion of embryonic day 6to embryonic day 14 of a mouse embryo, to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation mouse host embryo; and c) gestating the mouse hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric mouse having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation mouse host embryo incapable of developing endogenousgametes wherein the preimplantation mouse host embryo includes arecombinase operably linked to a developmentally regulated promoteractive in germ cells of the embryo during a portion of embryonic day 6to embryonic day 14 of a mouse embryo, to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation mouse host embryo; and c) gestating the mouse hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric mouse having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rat host embryo incapable of developing endogenousgametes wherein the preimplantation rat host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during a portion ofembryonic day 7 to embryonic day 16 of a rat embryo, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation rat host embryo; and c) gestating the rat hostembryo of b) under conditions suitable for development of the embryo,thereby generating a chimeric rat having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rat host embryo incapable of developing endogenousgametes wherein the preimplantation rat host embryo includes atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during a portion of embryonic day 7to embryonic day 16 of a rat embryo, to ablate the endogenous germ cellsof the embryo; b) introducing donor stem cells into the preimplantationrat host embryo; and c) gestating the rat host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric rat having substantially all gametes and/or germ cells derivedfrom the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation rat host embryo incapable of developing endogenousgametes wherein the preimplantation rat host embryo includes arecombinase operably linked to a developmentally regulated promoteractive in germ cells of the embryo during a portion of embryonic day 7to embryonic day 16 of a rat embryo, to ablate the endogenous germ cellsof the embryo; b) introducing donor stem cells into the preimplantationrat host embryo; and c) gestating the rat host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric rat having substantially all gametes and/or germ cells derivedfrom the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expression of a transgene, the transgene including a nucleicacid sequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further including at least one cytotoxic protein inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further includes a second transgeneincluding a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a promoterselected from the group consisting of: a developmentally regulatedpromoter and a ubiquitous promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter; b) introducing donor stem cells into the preimplantationnon-human host embryo; and c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expression of a transgene, the transgene including a nucleicacid sequence encoding an inhibitory RNA operably linked to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further including at least one inhibitory RNA inhibitorysequence and operably linked to at least two recombinase recognitionsites; wherein the non-human host embryo further includes a secondtransgene including a nucleic acid sequence encoding a recombinase, thenucleic acid sequence encoding the recombinase operably linked to apromoter selected from the group consisting of: a developmentallyregulated promoter and a ubiquitous promoter, wherein at least eitherthe first or second transgene is operably linked to a developmentallyregulated promoter; b) introducing donor stem cells into thepreimplantation non-human host embryo; and c) gestating the non-humanhost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein, an inhibitory RNA or a recombinase, wherein thenucleic acid sequence encodes a cytotoxic protein, an inhibitory RNA ora recombinase operably linked to an inducible promoter, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; and c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal having 80% ormore gametes and/or germ cells of the chimeric non-human animal arederived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expression of a first transgene, the first transgeneincluding a nucleic acid sequence encoding a cytotoxic protein operablylinked to a ubiquitous promoter or a developmentally regulated promoterselected from the group consisting of: vasa promoter, Dnd1 promoter,Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter, the transgene further including atleast one cytotoxic protein inhibitory sequence and operably linked toat least two recombinase recognition sites; wherein the non-human hostembryo further includes a second transgene including a nucleic acidsequence encoding a recombinase, the nucleic acid sequence encoding therecombinase operably linked to a ubiquitous promoter or adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2 promoter,Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiar promoter,wherein at least either the first or second transgene is operably linkedto a developmentally regulated promoter; b) introducing donor stem cellsinto the preimplantation non-human host embryo; and c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expression of a first transgene, the first transgeneincluding a nucleic acid sequence encoding an inhibitory RNA operablylinked to a ubiquitous promoter or a developmentally regulated promoterselected from the group consisting of: vasa promoter, c-kit promoter,Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter,Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter,Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAP promoter, thetransgene further including at least one cytotoxic protein inhibitorysequence and operably linked to at least two recombinase recognitionsites; wherein the non-human host embryo further includes a secondtransgene including a nucleic acid sequence encoding a recombinase, thenucleic acid sequence encoding the recombinase operably linked to aubiquitous promoter or a developmentally regulated promoter selectedfrom the group consisting of: vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter and TNAP promoter, wherein atleast either the first or second transgene is operably linked to adevelopmentally regulated promoter; b) introducing donor stem cells intothe preimplantation non-human host embryo; and c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice selected from the group consisting of: vasa promoter, Dnd1promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1promoter, Tex13 promoter, and Tiar promoter, to ablate the endogenousgerm cells of the embryo; b) introducing donor stem cells into thepreimplantation non-human host embryo; and c) gestating the non-humanhost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14in mice selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter, to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation non-human hostembryo; and c) gestating the non-human host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric non-human animal having substantially all gametes and/or germcells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes arecombinase linked to a developmentally regulated promoter active ingerm cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 in miceselected from the group consisting of: vasa promoter, c-kit promoter,Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter,Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter,Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAP promoter, toablate the endogenous germ cells of the embryo; b) introducing donorstem cells into the preimplantation non-human host embryo; and c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include, optionally, donorstem cells derived from a first animal species and a preimplantationnon-human host embryo of a different second animal species. In oneoption, the donor stem cells are rat stem cells and the preimplantationnon-human host embryo is a mouse embryo, such that rat gametes and/orrat germ cells are produced in a mouse host animal.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encodingHerpes simplex virus thymidine kinase operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring at least a portion of a developmental stage corresponding toembryonic day 6 to embryonic day 14 in mice and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation non-human host embryo; and c) gestating the non-humanhost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of endogenous gameteswherein the preimplantation non-human host embryo includes a transgene,the transgene including a nucleic acid sequence encoding a functionalmutant or truncated Herpes simplex virus thymidine kinase operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice and furthercomprising contacting endogenous germ cells expressing the a functionalmutant or truncated Herpes simplex virus thymidine kinase with athymidine analog to ablate the endogenous germ cells of the embryo; b)introducing donor stem cells into the preimplantation non-human hostembryo; and c) gestating the non-human host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric non-human animal having substantially all gametes and/or germcells derived from the donor stem cells.

Methods of producing a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encodingHerpes simplex virus thymidine kinase operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring at least a portion of a developmental stage corresponding toembryonic day 6 to embryonic day 14 in mice selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter, and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog to ablate the endogenous germcells of the embryo; b) introducing donor stem cells into thepreimplantation non-human host embryo; and c) gestating the non-humanhost embryo of b) under conditions suitable for development of theembryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes wherein the preimplantation non-human host embryo includes atransgene, the transgene including a nucleic acid sequence encodingHerpes simplex virus thymidine kinase operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring at least a portion of a developmental stage corresponding toembryonic day 6 to embryonic day 14 in mice selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter, and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog selected from the groupconsisting of: ganciclovir, acyclovir and fialuridine, to ablate theendogenous germ cells of the embryo; b) introducing donor stem cellsinto the preimplantation non-human host embryo; and c) gestating thenon-human host embryo of b) under conditions suitable for development ofthe embryo, thereby generating a chimeric non-human animal havingsubstantially all gametes and/or germ cells derived from the donor stemcells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expressing diphtheria toxin A fragment, attenuated diphtheriatoxin A fragment, tox-176; or a cytotoxic homologue, fragment or variantthereof, in germ cells of the non-human host embryo to ablate endogenousgerm cells; b) introducing donor stem cells into the preimplantationnon-human host embryo; and c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells.

Methods of generating a chimeric non-human animal are provided accordingto embodiments of the present invention which include: a) generating apreimplantation non-human host embryo incapable of developing endogenousgametes by expression of a first transgene, the first transgeneincluding a nucleic acid sequence encoding diphtheria toxin A fragment,attenuated DTA or tox-176; or a cytotoxic homologue, fragment or variantthereof, operably linked to a ubiquitous promoter or a developmentallyregulated promoter selected from the group consisting of: vasa promoter,Dnd1 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter,GDF-3 promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter,Prdm1 promoter, Tex13 promoter, and Tiar promoter, the transgene furtherincluding at least one inhibitory sequence for diphtheria toxin Afragment, attenuated DTA, tox-176 or a cytotoxic homologue, fragment orvariant thereof, and operably linked to at least two recombinaserecognition sites; wherein the non-human host embryo further includes asecond transgene including a nucleic acid sequence encoding arecombinase, the nucleic acid sequence encoding the recombinase operablylinked to a ubiquitous promoter or a developmentally regulated promoterselected from the group consisting of: vasa promoter, Dnd1 promoter,Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter; b) introducing donor stem cells into the preimplantationnon-human host embryo; and c) gestating the non-human host embryo of b)under conditions suitable for development of the embryo, therebygenerating a chimeric non-human animal having substantially all gametesand/or germ cells derived from the donor stem cells.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene encoding a deleter gene, thetransgene configured to express a cytotoxic protein or inhibitory RNA inendogenous germ cells of the embryo.

Non-human host embryos are provided according to embodiments of thepresent invention which include a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 of a mouse embryo.

Non-human host embryos are provided according to embodiments of thepresent invention which include a nucleic acid sequence encoding aninhibitory RNA operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14of a mouse embryo.

Host rodent embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding a cytotoxicprotein operably linked to a developmentally regulated promoter activein germ cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.

Host rodent embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding an inhibitoryRNA operably linked to a developmentally regulated promoter active ingerm cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.

Host mouse embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding a cytotoxicprotein operably linked to a developmentally regulated promoter activein germ cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.

Host mouse embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding an inhibitoryRNA operably linked to a developmentally regulated promoter active ingerm cells of the embryo during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.

Host rat embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding a cytotoxicprotein operably linked to a developmentally regulated promoter activein germ cells of the embryo during at least a portion of embryonic day 7to embryonic day 16 of the rat embryo.

Host rat embryos are provided according to embodiments of the presentinvention which include a nucleic acid sequence encoding an inhibitoryRNA operably linked to a developmentally regulated promoter active ingerm cells of the embryo during at least a portion of embryonic day 7 toembryonic day 16 of the rat embryo.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further including at least one cytotoxic protein inhibitorysequence and operably linked to at least two recombinase recognitionsites; wherein the non-human host embryo further includes a secondtransgene including a nucleic acid sequence encoding a recombinase, thenucleic acid sequence encoding the recombinase operably linked to apromoter selected from the group consisting of: a developmentallyregulated promoter and a ubiquitous promoter, wherein at least eitherthe first or second transgene is operably linked to a developmentallyregulated promoter.

Non-human host embryos are provided according to embodiments of thepresent invention which include a first transgene including a nucleicacid sequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2 promoter,Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiar promoter, or aubiquitous promoter, the first transgene further including at least onecytotoxic protein inhibitory sequence and operably linked to at leasttwo recombinase recognition sites; wherein the non-human host embryofurther includes a second transgene including a nucleic acid sequenceencoding a recombinase, the nucleic acid sequence encoding therecombinase operably linked to a developmentally regulated promoterselected from the group consisting of: vasa promoter, Dnd1 promoter,Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter, or a ubiquitous promoter, wherein atleast either the first or second transgene is operably linked to adevelopmentally regulated promoter.

Non-human host embryos are provided according to embodiments of thepresent invention which include a first transgene including a nucleicacid sequence encoding an inhibitory RNA operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter, or a ubiquitous promoter, the firsttransgene further including at least one inhibitory RNA inhibitorysequence and operably linked to at least two recombinase recognitionsites; wherein the non-human host embryo further includes a secondtransgene including a nucleic acid sequence encoding a recombinase, thenucleic acid sequence encoding the recombinase operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter, or a ubiquitous promoter, wherein at leasteither the first or second transgene is operably linked to adevelopmentally regulated promoter.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to an induciblepromoter or a ubiquitous promoter, the transgene further including atleast one cytotoxic protein inhibitory sequence and operably linked toat least two recombinase recognition sites; wherein the non-human hostembryo further includes a second transgene including a nucleic acidsequence encoding a recombinase, the nucleic acid sequence encoding therecombinase operably linked to a promoter selected from the groupconsisting of: an inducible promoter and a ubiquitous promoter, whereinat least either the first or second transgene is operably linked to aninducible promoter.

Non-human host embryos are provided according to embodiments of thepresent invention which include a nucleic acid sequence encoding acytotoxic protein operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 of a mouse embryo selected from vasa promoter, Dnd1 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, or Tiar promoter.

Non-human host embryos are provided according to embodiments of thepresent invention which are incapable of developing endogenous gametesdue to ablation of germ cells as described herein. The non-human hostincapable of developing endogenous gametes due to ablation of germ cellscan be embryos of any non-human mammal including, but not limited to,any rodent, mouse, rat non-human primate, marmoset, rabbit, dog, cat,cattle, horse, sheep, goat, an endangered mammal or an exotic mammal.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene which includes a nucleicacid sequence encoding a Herpes simplex virus thymidine kinase operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryo.The endogenous germ cells expressing the Herpes simplex virus thymidinekinase are contacted with a thymidine analog to ablate the endogenousgerm cells.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene which includes a nucleicacid sequence encoding a diphtheria toxin A fragment, attenuated DTA,tox-176 or a cytotoxic homologue, fragment or variant thereof, operablylinked to vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2promoter, Nanos3 promoter, Prdm1 promoter, Tex13 promoter, or Tiarpromoter. The endogenous germ cells expressing the Herpes simplex virusthymidine kinase are contacted with a thymidine analog to ablate theendogenous germ cells.

Non-human host embryos are provided according to embodiments of thepresent invention which include a transgene which includes a nucleicacid sequence encoding a Herpes simplex virus thymidine kinase operablylinked to vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter,Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter or TNAP promoter. The endogenous germ cells expressing theHerpes simplex virus thymidine kinase are contacted with a thymidineanalog to ablate the endogenous germ cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene encoding a cytotoxicprotein or RNA interference molecule into the embryo; and expressing thetransgene in endogenous germ cells of the embryo, thereby producing anon-human embryo lacking functional endogenous germ cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter active in germ cells of the embryoduring at least a portion of a developmental stage corresponding toembryonic day 6 to embryonic day 14 in mice. The transgene is expressedin endogenous germ cells of the embryo, thereby producing a non-humanembryo lacking functional endogenous germ cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter active in germ cells of the embryo during at least aportion of a developmental stage corresponding to embryonic day 6 toembryonic day 14 in mice. The transgene is expressed in endogenous germcells of the embryo, thereby producing a non-human embryo lackingfunctional endogenous germ cells.

Methods of producing a host rodent embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter active in germ cells of the rodentembryo during at least a portion of a developmental stage correspondingto embryonic day 6 to embryonic day 14 in mice. The transgene isexpressed in endogenous germ cells of the rodent embryo, therebyproducing a rodent embryo lacking functional endogenous germ cells.

Methods of producing a host rodent embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter active in germ cells of the rodent embryo during atleast a portion of a developmental stage corresponding to embryonic day6 to embryonic day 14 in mice. The transgene is expressed in endogenousgerm cells of the rodent embryo, thereby producing a rodent embryolacking functional endogenous germ cells.

Methods of producing a mouse embryo incapable of developing endogenousgametes are provided according to embodiments of the present inventionwhich include introducing a transgene including a nucleic acid sequenceencoding a cytotoxic protein operably linked to a developmentallyregulated promoter active in germ cells of the mouse embryo during atleast a portion of a developmental stage corresponding to embryonic day6 to embryonic day 14 in mice. The transgene is expressed in endogenousgerm cells of the mouse embryo, thereby producing a mouse embryo lackingfunctional endogenous germ cells.

Methods of producing a host mouse embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter active in germ cells of the mouse embryo during atleast a portion of a developmental stage corresponding to embryonic day6 to embryonic day 14 in mice. The transgene is expressed in endogenousgerm cells of the mouse embryo, thereby producing a mouse embryo lackingfunctional endogenous germ cells.

Methods of producing a rat rodent embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter active in germ cells of the ratembryo during at least a portion of embryonic day 7 to embryonic day 16of the rat embryo. The transgene is expressed in endogenous germ cellsof the rat embryo, thereby producing a rat embryo lacking functionalendogenous germ cells.

Methods of producing a host rat embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter active in germ cells of the rat embryo during atleast a portion of embryonic day 7 to embryonic day 16 of the ratembryo. The transgene is expressed in endogenous germ cells of the ratembryo, thereby producing a rat embryo lacking functional endogenousgerm cells.

Methods of producing a mouse embryo incapable of developing endogenousgametes are provided according to embodiments of the present inventionwhich include introducing a first transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further including at least one cytotoxic protein inhibitorysequence and operably linked to at least two recombinase recognitionsites; wherein the non-human host embryo further comprises a secondtransgene comprising a nucleic acid sequence encoding a recombinase, thenucleic acid sequence encoding the recombinase operably linked to apromoter selected from the group consisting of: a developmentallyregulated promoter and a ubiquitous promoter, wherein at least eitherthe first or second transgene is operably linked to a developmentallyregulated promoter.

Methods of producing a mouse embryo incapable of developing endogenousgametes are provided according to embodiments of the present inventionwhich include introducing a first transgene including a nucleic acidsequence encoding a cytotoxic protein operably linked to adevelopmentally regulated promoter selected from vasa promoter, Dnd1promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1promoter, Tex13 promoter, and Tiar promoter, or a ubiquitous promoter,the transgene further including at least one cytotoxic proteininhibitory sequence and operably linked to at least two recombinaserecognition sites; wherein the non-human host embryo further comprises asecond transgene comprising a nucleic acid sequence encoding arecombinase, the nucleic acid sequence encoding the recombinase operablylinked to a developmentally regulated promoter selected from vasapromoter, Dnd1 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanos2 promoter, Nanos3promoter, Prdm1 promoter, Tex13 promoter, and Tiar promoter, and aubiquitous promoter, wherein at least either the first or secondtransgene is operably linked to a developmentally regulated promoter.

Methods of producing a mouse embryo incapable of developing endogenousgametes are provided according to embodiments of the present inventionwhich include introducing a first transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter or a ubiquitous promoter, the transgene furtherincluding at least one inhibitory RNA inhibitory sequence and operablylinked to at least two recombinase recognition sites; wherein thenon-human host embryo further comprises a second transgene comprising anucleic acid sequence encoding a recombinase, the nucleic acid sequenceencoding the recombinase operably linked to a promoter selected from thegroup consisting of: a developmentally regulated promoter and aubiquitous promoter, wherein at least either the first or secondtransgene is operably linked to a developmentally regulated promoter.

Methods of producing a mouse embryo incapable of developing endogenousgametes are provided according to embodiments of the present inventionwhich include introducing a first transgene including a nucleic acidsequence encoding an inhibitory RNA operably linked to a developmentallyregulated promoter selected from vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct314 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter and TNAP promoter, or aubiquitous promoter, the transgene further including at least oneinhibitory RNA inhibitory sequence and operably linked to at least tworecombinase recognition sites; wherein the non-human host embryo furthercomprises a second transgene comprising a nucleic acid sequence encodinga recombinase, the nucleic acid sequence encoding the recombinaseoperably linked to a developmentally regulated promoter selected fromvasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter, or a ubiquitous promoter, wherein at leasteither the first or second transgene is operably linked to adevelopmentally regulated promoter.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding a cytotoxic protein or RNA interference molecule intothe embryo, the nucleic acid sequence operably linked to an induciblepromoter; and expressing, the transgene in endogenous germ cells of theembryo, thereby producing a non-human embryo lacking functionalendogenous germ cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene including a nucleic acidsequence encoding a cytotoxic protein or an inhibitory RNA, the nucleicacid sequence encoding the cytotoxic protein or the inhibitory RNAoperably linked to a developmentally regulated promoter active in germcells of the embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice selectedfrom the group consisting of: vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter and TNAP promoter.

Methods of producing a non-human host embryo lacking endogenous germcells are provided according to embodiments of the present inventionwhich include introducing a transgene encoding Herpes simplex virusthymidine kinase into the embryo; expressing the transgene in endogenousgerm cells of the embryo, thereby producing a non-human embryo lackingfunctional endogenous germ cells; contacting endogenous germ cellsexpressing the Herpes simplex virus thymidine kinase with a thymidineanalog to ablate the endogenous germ cells.

Methods of producing a non-human host embryo lacking endogenous germcells are provided according to embodiments of the present inventionwhich include introducing a transgene encoding Herpes simplex virusthymidine kinase operably linked to a developmentally regulated promoterinto the embryo; expressing the transgene in endogenous germ cells ofthe embryo, thereby producing a non-human embryo lacking functionalendogenous germ cells; contacting endogenous germ cells expressing theHerpes simplex virus thymidine kinase with ganciclovir, acyclovir orfialuridine to ablate the endogenous germ cells.

A combination of any two or more thymidine analogs, such as acombination of any two or more of ganciclovir, acyclovir andfialuridine, can be used.

Methods of producing a non-human host embryo lacking endogenous germcells are provided according to embodiments of the present inventionwhich include introducing a transgene encoding Herpes simplex virusthymidine kinase operably linked vasa promoter, c-kit promoter, Dnd1promoter, Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2promoter, GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2promoter, Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14promoter, Tex13 promoter, Tiar promoter or TNAP promoter, into theembryo; expressing the transgene in endogenous germ cells of the embryo,thereby producing a non-human embryo lacking functional endogenous germcells; and contacting endogenous germ cells expressing the Herpessimplex virus thymidine kinase with ganciclovir, acyclovir orfialuridine to ablate the endogenous germ cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include introducing a transgene encoding diphtheriatoxin A fragment, attenuated DTA, tox-176, or a cytotoxic homologue,fragment or variant thereof, operably linked to vasa promoter, Dnd1promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1promoter, Tex13 promoter, or Tiar promoter, into the embryo; andexpressing the transgene in endogenous germ cells of the embryo, therebyproducing a non-human embryo lacking functional endogenous germ cells.

Non-human host embryos incapable of developing endogenous gametes areprovided according to embodiments of the present invention, thenon-human host embryos including a transgene encoding a recombinaseoperably linked to a developmentally regulated promoter active in germcells and a transgene encoding diphtheria toxin operably linked to aubiquitous promoter, wherein the transgene encoding diphtheria toxinoperably linked to a ubiquitous promoter having a loxP-flanked stopcassette and operably linked to at least two recombinase recognitionsites.

Non-human host embryos incapable of developing endogenous gametes areprovided according to embodiments of the present invention, thenon-human host embryos including a first transgene encoding arecombinase operably linked to a developmentally regulated promoteractive in germ cells selected from vasa promoter, Dnd1 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter,Tex13 promoter, and Tiar promoter; and a second transgene encoding: aloxP-flanked stop cassette operably linked to a nucleic acid sequenceencoding diphtheria toxin A fragment, attenuated DTA, tox-176, or acytotoxic homologue, fragment or variant thereof. The nucleic acidsequence encoding diphtheria toxin A fragment, attenuated DTA, tox-176,or a cytotoxic homologue, fragment or variant thereof is operably linkedto a ubiquitous promoter. The loxP-flanked stop cassette is operablylinked to at least two recombinase recognition sites such thatexpression of the recombinase is effective to excise the stop cassettesuch that the diphtheria toxin A fragment, attenuated DTA, tox-176, or acytotoxic homologue, fragment or variant thereof operably linked to aubiquitous promoter is expressed in the germ cells, thereby ablating thegerm cells.

Non-human host embryos incapable of developing endogenous gametes areprovided according to embodiments of the present invention, thenon-human host embryos including a transgene encoding a recombinaseoperably linked to a vasa promoter and a transgene encoding diphtheriatoxin operably linked to a ubiquitous promoter, wherein the transgeneencoding diphtheria toxin is operably linked to a ubiquitous promoterhaving a loxP-flanked stop cassette and operably linked to at least tworecombinase recognition sites.

Non-human host embryos incapable of developing endogenous gametes areprovided according to embodiments of the present invention, thenon-human host embryos including a first transgene encoding arecombinase operably linked to a vasa promoter; and a second transgeneencoding: a loxP-flanked stop cassette operably linked to a nucleic acidsequence encoding diphtheria toxin A fragment, attenuated DTA, tox-176,or a cytotoxic homologue, fragment or variant thereof. The nucleic acidsequence encoding diphtheria toxin A fragment, attenuated DTA, tox-176,or a cytotoxic homologue, fragment or variant thereof is operably linkedto a ubiquitous promoter. The loxP-flanked stop cassette is operablylinked to at least two recombinase recognition sites such thatexpression of the recombinase is effective to excise the stop cassettesuch that the diphtheria toxin A fragment, attenuated DTA, tox-176, or acytotoxic homologue, fragment or variant thereof operably linked to aubiquitous promoter is expressed in the germ cells, thereby ablating thegerm cells.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include breeding a first animal of a first rodent straincomprising a transgene encoding a recombinase operably linked to adevelopmentally regulated promoter and a second animal of a secondrodent strain carrying a transgene with recombinase recognition sitesoperably linked to a nucleic acid sequence encoding a cytotoxic proteinor inhibitory RNA operably linked to a ubiquitous or developmentallyregulated promoter.

Methods of producing a non-human host embryo incapable of developingendogenous gametes are provided according to embodiments of the presentinvention which include breeding a first mouse strain comprising atransgene encoding Cre recombinase operably linked with a vasa promoterand a second mouse strain comprising a loxP-flanked stop cassetteoperatively linked with a transgene encoding diphtheria toxin, such asdiphtheria toxin A fragment, attenuated DTA, tox-176, or a cytotoxichomologue, fragment or variant thereof, operably linked with aubiquitous or developmentally regulated promoter.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of a DNA expression constructcontaining a site specific recombinase gene to create an activator mousestrain; sequence lengths are not drawn to scale;

FIG. 2 is a schematic representation of a DNA expression constructcontaining a site specific recombinase gene to create an activator mousestrain; sequence lengths are not drawn to scale;

FIG. 3 is a schematic representation of a DNA expression construct usedto create a deleter mouse strain; sequence lengths are not drawn toscale;

FIG. 4 is a schematic representation of a shRNA or RNAi expressionconstruct; sequence lengths are not drawn to scale;

FIG. 5 is a schematic representation of an inducible DNA expressionconstruct; sequence lengths are not drawn to scale;

FIG. 6 is a schematic representation of a shRNA or RNAi expressionconstruct; sequence lengths are not drawn to scale;

FIGS. 7A and 7B are images of photomicrographs of stained sections oftestes derived from normal mice and F1 animals fromVasa-Cre×ROSA26-eGFP-DTA (deleter) crosses;

FIG. 8 is an image of a photomicrograph of testes from chimeras;

FIG. 9A is an image of a photomicrograph of stained sections of testesfrom infertile chimera #2;

FIG. 9B is an image of a photomicrograph of stained sections of testesfrom fertile chimera #3;

FIG. 9C is an image of a photomicrograph of stained sections of testesfrom wild type control testis;

FIG. 10 is an image of a photomicrograph of testes from wild typecontrol and F1 animals from Vasa-Cre×HoxD<tm1Kmta> mice; and

FIG. 11 is a schematic representation of a DNA construct used to createa deleter mouse strain by integrating two loxP sites in inverseorientation; sequence lengths are not drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods are provided according to the present inventionrelating to non-human animals modified to promote production of selectedgerm cells and gametes from exogenous stem cells.

Modified non-human host embryos and methods for producing the modifiednon-human host embryos are provided by the present invention. Germ cellsoriginating in the non-human host embryos are manipulated such that theyare not capable of proliferation and/or differentiation into gametes.Modified non-human host embryos are used to “host” introduced donor stemcells which populate the germ cell layer, resulting in chimericnon-human animals in which the germ cells and gametes are all orsubstantially all from donor stem cells.

Methods and compositions are provided according to embodiments of thepresent invention for generation of chimeric non-human animals in whichthe germ cells and/or gametes are all or substantially all from donorstem cells. Thus, methods and compositions are provided for generationof chimeric non-human animals in which the germ cells and/or gametes are80% or greater derived from donor stem cells.

Methods of generating a chimeric non-human embryo or animal in which thegerm cells and/or gametes are all or substantially all derived fromdonor stem cells include generating a non-human host embryo lackingfunctional germ cells. Such methods further include introduction ofdonor stem cells into the non-human host embryo before ablation of thegerm cells endogenous to the non-human host embryo. The non-human hostembryo lacking functional endogenous germ cells and including introduceddonor stem cells is gestated under conditions suitable for developmentof the embryo, thereby generating a chimeric non-human animal havingsubstantially all germ cells and/or gametes derived from the donor stemcells. The germ cells and gametes can then be used to make a non-humanembryo or animal derived from the donor stem cells.

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company,2004; Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAiTechnology, DNA Press LLC, Eagleville, Pa., 2003; Herdewijn, P. (Ed.),Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K.Vintersten, R. Behringer (Eds) 2002, Manipulating the Mouse Embryo: ALaboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press,ISBN-10: 0879695919; K. Turksen (Ed.), Embryonic stem cells: methods andprotocols in Methods Mol. Biol. 2002; 185, Humana Press; CurrentProtocols in Stem Cell Biology, ISBN: 9780470151808.

The singular terms “a,” “an,” and “the” are not intended to be limitingand include plural referents unless explicitly state or the contextclearly indicates otherwise.

The terms “germ cell” and “germ cells” are used interchangeably andrefer to cells that give rise to gametes. The term “germ cells” includesprimordial germ cells, cells positive for alkaline phosphatase, primaryoocytes, oogonia, spermatogonial stem cells, spermatogonia and primaryspermatocytes.

The terms “gamete” and “gametes” are used interchangeably and refer tosecondary germ cells, including oocytes, ova, spermatozoa and sperm.

The term “breeding” as used herein, means the union of male and femalegametes so that fertilization occurs. Such a union may be brought aboutby mating (copulation) or by in vitro or in vivo artificial methods.Such artificial methods include, but are not limited to, artificialinsemination, surgical assisted artificial insemination, in vitrofertilization, intracytoplasmic sperm injection, zona drilling, in vitroculture of fertilized oocytes, ovary transfer and ovary splitting.

The term “transgenic” as used herein refers to a genetically modifiednon-human animal containing a transgene. The term “transgene” as usedherein refers to a nucleic acid artificially inserted into the genome ofa non-human animal, transiently, or more preferably, permanentlyintroducing a genetic change in the non-human animal.

The term “genetically modified” as used herein refers to theintroduction of DNA technology into a cell or organism.

Compositions and methods of the present invention are not limited toparticular amino acid and nucleic sequences identified by SEQ ID NOherein and homologues and variants of a reference nucleic acid orprotein may be used.

Homologues and variants of a nucleic acid or protein described hereinare characterized by conserved functional properties compared to thecorresponding nucleic acid or protein.

Percent identity is determined by comparison of amino acid or nucleicacid sequences, including a reference amino acid or nucleic acidsequence and a putative homologue amino acid or nucleic acid sequence.To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Thetwo sequences compared are generally the same length or nearly the samelength.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. Algorithms used fordetermination of percent identity illustratively include the algorithmsof S. Karlin and S. Altshul, PNAS, 90:5873-5877, 1993; T. Smith and M.Waterman, Adv. Appl. Math. 2:482-489, 1981, S, Needleman and C. Wunsch,J. Mol. Biol., 48:443-453, 1970, W. Pearson and D. Lipman, PNAS,85:2444-2448, 1988 and others incorporated into computerizedimplementations such as, but not limited to, GAP, BESTFIT, FASTA,TFASTA; and BLAST, for example incorporated in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.) and publicly available from the National Center for BiotechnologyInformation.

A non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,1990, PNAS 87:2264-2268, modified as in Karlin and Altschul, 1993, PNAS.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches are performed with the NBLAST nucleotide programparameters set, e.g., for score=100, word length=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentinvention. BLAST protein searches are performed with the XBLAST programparameters set, e.g., to score 50, word length=3 to obtain amino acidsequences homologous to a protein molecule of the present invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST areutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI BLAST is used to perform an iteratedsearch which detects distant relationships between molecules. Whenutilizing BLAST, Gapped BLAST, and PSI Blast programs, the defaultparameters of the respective programs (e.g., of XBLAST and NBLAST) areused. Another preferred, non limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, 1988, CABIOS 4:11-17. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 is used.

The percent identity between two sequences is determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

One of skill in the art will recognize that one or more nucleic acid oramino acid mutations can be introduced without altering the functionalproperties of a given nucleic acid or protein, respectively. Mutationscan be introduced using standard molecular biology techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis, to producevariants. For example, one or more amino acid substitutions, additions,or deletions can be made without altering the functional properties of areference protein. Similarly, one or more nucleic acid substitutions,additions, or deletions can be made without altering the functionalproperties of a reference promoter sequence.

When comparing a reference protein to a putative homologue, amino acidsimilarity may be considered in addition to identity of amino acids atcorresponding positions in an amino acid sequence. “Amino acidsimilarity” refers to amino acid identity and conservative amino acidsubstitutions in a putative homologue compared to the correspondingamino acid positions in a reference protein.

Conservative amino acid substitutions can be made in reference proteinsto produce variants.

Conservative amino acid substitutions are art recognized substitutionsof one amino acid for another amino acid having similar characteristics.For example, each amino acid may be described as having one or more ofthe following characteristics: electropositive, electronegative,aliphatic, aromatic, polar, hydrophobic and hydrophilic. A conservativesubstitution is a substitution of one amino acid having a specifiedstructural or functional characteristic for another amino acid havingthe same characteristic. Acidic amino acids include aspartate,glutamate; basic amino acids include histidine, lysine, arginine;aliphatic amino acids include isoleucine, leucine and valine; aromaticamino acids include phenylalanine, glycine, tyrosine and tryptophan;polar amino acids include aspartate, glutamate, histidine, lysine,asparagine, glutamine, arginine, serine, threonine and tyrosine; andhydrophobic amino acids include alanine, cysteine, phenylalanine,glycine, isoleucine, leucine, methionine, proline, valine andtryptophan; and conservative substitutions include substitution amongamino acids within each group. Amino acids may also be described interms of relative size, alanine, cysteine, aspartate, glycine,asparagine, proline, threonine, serine, valine, all typically consideredto be small.

A variant can include synthetic amino acid analogs, amino acidderivatives and/or non-standard amino acids, illustratively including,without limitation, alpha-aminobutyric acid, citrulline, canavanine,cyanoalanine, diaminobutyric acid, diaminopimelic acid,dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline,norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan,1-methylhistidine, 3-methylhistidine, and ornithine.

With regard to nucleic acids, it will be appreciated by those of skillin the art that due to the degenerate nature of the genetic code,multiple nucleic acid sequences can encode a particular protein, andthat such alternate nucleic acids may be used in compositions andmethods of the present invention.

The term “expression construct” is used herein to refer to adouble-stranded recombinant DNA molecule containing a nucleic acidsequence desired to be expressed and containing appropriate regulatoryelements necessary or desirable for the transcription of the operablylinked nucleic acid sequence in vitro or in vivo. The term “recombinant”is used to indicate a nucleic acid construct in which two or morenucleic acids are linked and which are not found linked in nature. Theterm “nucleic acid” as used herein refers to RNA or DNA molecules havingmore than one nucleotide in any form including single-stranded,double-stranded, oligonucleotide or polynucleotide. The term “nucleotidesequence” is used to refer to the ordering of nucleotides in anoligonucleotide or polynucleotide in a single-stranded form of nucleicacid. The term “expressed” refers to transcription of a nucleic acidsequence to produce a corresponding mRNA and/or translation of the mRNAto produce the corresponding protein. Expression constructs can begenerated recombinantly or synthetically or by DNA synthesis usingwell-known methodology.

An expression construct is introduced into a cell using well-knownmethodology, such as, but not limited to, by introduction of a vectorcontaining the expression construct into the cell. A “vector” is anucleic acid molecule that transfers an inserted nucleic acid moleculeinto and/or between host cells becoming self-replicating. The termincludes vectors that function primarily for insertion of a nucleic acidmolecule into a cell, replication of vectors that function primarily forthe replication of nucleic acid, and expression vectors that functionfor transcription and/or translation of the DNA or RNA. Also includedare vectors that provide more than one of the above functions.

Vectors include plasmids, viruses, BACs, YACs, and the like. Particularviral vectors illustratively include those derived from adenovirus,adeno-associated virus and lentivirus.

Any of various methods can be used to introduce a transgene into anon-human animal to produce a transgenic animal. Such techniques arewell-known in the art and include, but are not limited to, pronuclearmicroinjection, viral infection and transformation of embryonic stemcells and iPS cells. Methods for generating transgenic animals that canbe used include, but are not limited to, those described in J. P.Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRCPress; 2006; M. H. Hofker and I. van Deursen, Eds., Transgenic MouseMethods and Protocols, Humana Press, 2002; A. L. Joyner, Gene Targeting:A Practical Approach, Oxford University Press, 2000; Manipulating theMouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press; 2002, ISBN-10: 0879695919; K. Turksen (Ed.), Embryonicstem cells: methods and protocols in Methods Mol. Biol. 2002; 185,Humana Press; Current Protocols in Stem Cell Biology, ISBN:978047015180; Meyer et al. PNAS USA, vol. 107 (34), 15022-15026.

The term “regulatory element” as used herein refers to a nucleotidesequence which controls some aspect of the expression of an operablylinked nucleic acid sequence. Exemplary regulatory elementsillustratively include an enhancer, an internal ribosome entry site(IRES), an intron; an origin of replication, a polyadenylation signal(pA), a promoter, a transcription termination sequence, and an upstreamregulatory domain, which contribute to the replication, transcription,post-transcriptional processing of a nucleic acid sequence. Those ofordinary skill in the art are capable of selecting and using these andother regulatory elements in an expression construct with no more thanroutine experimentation.

The term “operably linked” as used herein refers to a nucleic acid infunctional relationship with a second nucleic acid. The term “operablylinked” encompasses functional connection of two or more nucleic acidmolecules, such as an oligonucleotide or polynucleotide to betranscribed and a regulatory element such as a promoter or an enhancerelement, which allows transcription of the oligonucleotide orpolynucleotide to be transcribed.

The term “promoter” as used herein refers to a DNA sequence operablylinked to a nucleic acid sequence to be transcribed such as a nucleicacid sequence encoding a desired molecule. A promoter is generallypositioned upstream of a nucleic acid sequence to be transcribed andprovides a site for specific binding by RNA polymerase and othertranscription factors. In specific embodiments, a promoter is generallypositioned upstream of the nucleic acid sequence transcribed to producethe desired molecule, and provides a site for specific binding by RNApolymerase and other transcription factors.

In addition to a promoter, one or more enhancer sequences may beincluded such as, but not limited to, cytomegalovirus (CMV) earlyenhancer element and an SV40 enhancer element. Additional includedsequences are an intron sequence such as the beta globin intron or ageneric intron, a transcription termination sequence, and an mRNApolyadenylation (pA) sequence such as, but not limited to SV40-pA,beta-globin-pA, the human growth hormone (hGH) pA and SCF-pA. The term“polyA” or “p(A)” or “pA” refers to nucleic acid sequences that signalfor transcription termination and mRNA polyadenylation. The polyAsequence is characterized by the hexanucleotide motif AAUAAA. Commonlyused polyadenylation signals are the SV40 pA, the human growth hormone(hGH) pA, the beta-actin pA, and beta-globin pA. The sequences can rangein length from 32 to 450 bp. Multiple pA signals may be used.

Optionally, a reporter gene is included in the transgene construct. Theterm “reporter gene” as used herein refers to gene that is easilydetectable when expressed, for example via chemiluminescence,fluorescence, colorimetric reactions, antibody binding, induciblemarkers, ligand binding assays, and the like. Exemplary reporter genesinclude but are not limited to green fluorescent protein (GFP; seeMistili and Spector, Nature Biotechnology 15:961-964 (1997), eGFP, YFP,eYFP, CFP, eCFP, BFP, eBFP, MmGFP, a modified GFP, dsRed (redfluorescent protein, RFP), luciferase and beta-galactosidase (lacZ).

As will be recognized by the skilled artisan, the 5′ non-coding regionof a gene can be isolated and used in its entirety as a promoter in atransgene to drive expression of an operably linked nucleic acid.Alternatively, a portion of the 5′ non-coding region can be isolated andinserted in a transgene to drive expression of an operably linkednucleic acid. In general, about 500-6000 bp of the 5′ non-coding regionof a developmentally regulated gene is included in a transgene to conferdevelopmentally regulated expression of the operably linked nucleic acidencoding a cytotoxic protein, inhibitory RNA or recombinase for germcell ablation and ablate germ cells. Optionally, a portion of the 5′non-coding region of a developmentally regulated gene containing aminimal amount of the 5′ non-coding region needed to conferdevelopmentally regulated expression of the operably linked nucleic acidencoding a cytotoxic protein, inhibitory RNA or recombinase for germcell ablation. Assays described herein can be used to determine theability of a designated portion of the 5′ non-coding region of adevelopmentally regulated gene to confer developmentally regulatedexpression of the operably linked nucleic acid encoding a cytotoxicprotein, inhibitory RNA or recombinase for germ cell ablation.

The term “developmentally regulated promoter” as used herein refers to apromoter that is active during at least a portion of embryonicdevelopment, in the case of mouse that is any embryonic day from day 6to embryonic day 14 (E6, E6.5, E7, E7.5 E8, E8.5, E9, E9.5, E10, E10.5,E11, E11.5, E12, E12.5, E13, E13.5 and E14), also conventionallydescribed as Theiler stages TS8, TS9, TS10, TS11, TS12, TS13, TS14,TS15, TS16, TS17, TS18, TS19, TS20, TS21, and TS22 and active inprimordial germ cells and/or during germ cell development and/or germcell differentiation. The developmentally regulated promoter is activeduring at least a portion of embryonic day 7.0 to embryonic day 10.5 (E7to E10.5), also conventionally described as Theiler stages TS10 to TS17in mouse. For rat the developmentally regulated promoter is activeduring at least a portion from embryonic day 7 to embryonic day 15.5(E7, E7.5 E8, E8.5, E9, E9.5, E10, E10.5, E11, E11.5, E12, E12.5, E13,E13.5, E14, E14.5, E15, E15.5 and E16), also conventionally described asWitschi stages 10 to 33. For other species, the developmentallyregulated promoter is active in a developmental stage corresponding tothose described above for mouse.

Promoters described herein are known to be active in primordial germcells and/or during germ cell development and/or germ celldifferentiation. Additional promoters useful in methods and compositionsof the present invention may be determined to be active in primordialgerm cells and/or during germ cell development and/or germ celldifferentiation using conventional techniques, such as analysis ofexpression of RNA or protein produced from a nucleic acid construct inwhich the promoter is operably linked to a nucleic acid encoding the RNAor protein in primordial germ cells and/or during germ cell developmentand/or germ cell differentiation.

Developmentally regulated promoters are known in the art, as exemplifiedherein.

Promoters active in the early stages of germ cell development arepreferred in order to create a niche in the host embryo to reduce thecompetition of the donor stein cells with host cells.

Developmentally regulated promoters include, but are not limited to vasasuch as mouse vasa (mouse vasa homologue, Mvh, Ddx4, DDX4) promoter asdescribed in Toyooka Y, et al., 2000, Mech Dev. 93(1-2):139-49 andEP1,911,842; human VASA promoter as described in Kee et al. 2009, Nature462, 222-225 and EP1,911,842; SEQ ID NO:1 and Table 3 for a listing ofrelevant sequence recognition sites for transcription factor bindingincluding SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; c-kitpromoter as described in Yasuda H et al, Biochem Biophys Res Commun1993; 191:893-901 and see SEQ ID NO 2; Dppa3 (also known as stella orPgc7) promoter as described in Hirota T et al 2011, Biol Reproduction 85(2), 367-377; daz1 (also known as dazh or DAZH) promoter as described inReijo R et al 1996 Genomics 35, 346-352; Linhera K et al 2009,Differentiation 77 (4), 335-349, for example a 1.7 kb promoter fragmentupstream of the translational start site of Daz1 promoter is sufficientfor tissue-specific expression (Nicholas et al 2009, Genesis 47, 74-84);Dnd1 (Dead end homolog 1) promoter as described in Youngren et al. 2005,Nature 435, 360-364 and Table 4 for a listing of relevant sequencerecognition sites for transcription factor binding and SEQ ID NO:7 toSEQ ID NO:15; Fkbp6 (FK506 binding protein 6 or Fkbp36) as described inMeng et al 1998, Genomics 52, 130-137 and Patterson et al 2002, Genomics79, 881-889 and Table 5 for a listing of relevant sequence recognitionsites for transcription factor binding and SEQ ID NO:16 to SEQ ID NO:24;Fragilis (mil-1, Ifitm3, interferon-induced transmembrane protein 3)promoter as described in Tanaka S S et al 2002, Mech Dev 1195,S261-S267; Tanaka S S et al 2004 Dev Dyn, 230:651-659 and Lange et al.,2003, BMC Dev Biol 3: 1; Fragilis-2 (mil-2, Ifitm1, interferon-inducedtransmembrane protein 1) promoter as described in Tanaka et al 2002,Mech Dev 119S, 5261-S267 and Lange et al 2003, BMC Dev Biol 3:1; GDF-3promoter as described in Clark A T, Stem Cells, 2004; 22(2): 169-79;Mov1011 (Mov10 like-1, a putative RNA helicase) as described in Wang etal. 2001, Nat Genet. 27, 422-426 and Table 6 for a listing of relevantsequence recognition sites for transcription factor binding and SEQ IDNO:25 to SEQ ID NO:31; Nanog promoter as described in Wu and Yao, 2005,Cell Res, 15(5):317-24; Nanos2 promoter as described in Suzuki et al.,2007, Development 134, 77-83; Nanos3 promoter as described in Suzuki etal 2008, Dev Biol 318, 133-142 and Suzuki et al., 2010, PLoS One,5(2):e9300; oct3/4 (also known as oct-4 or) promoter as described inYeom et al 1991, Mech Develop 35 (3), 171-179, Sylvester et al 1994,Nucleic Acids Res 22:901-911 and Nordhoff et al 2001, Mammalian Genome12 (4), 309-317 and SEQ ID NO:39; Prdm1 (Blimp-1) as described in Turneret al., 1994, Cell 77, 297-306 and Ohinata Y et al 2005, Nature436:207-213; Prdm14 promoter (a PR domain-containing transcriptionalregulator) as described in Yamaji M et al. 2008, Nature Genet. 40,1016-1022; Tex13 (testis-expressed gene 13) as described in Wang et al.2001, Nat Genet. 27, 422-426 and GenBank no. AF285576.1 and Table 7 fora listing of relevant sequence recognition sites for transcriptionfactor binding, and SEQ ID NO:32 to SEQ ID NO:34; Tiar (also known asTIAL1) promoter as described in Tominaga et al., 2010, Genes to Cells,15, Issue 6, 595-606; and TNAP (also known as Alp1, alkaline phosphataseliver, bone, kidney) promoter as described in MacGregor G R et al (1995)Development, 121 (5), 1487-1496 and Lomeli H et al 2000, Genesis26:116-117. Table 1 lists developmentally regulated promoters, and Table2 lists a set of more tightly developmentally regulated promoters.

A developmentally regulated promoter included in a transgene andoperably linked nucleic acid encoding a cytotoxic protein, inhibitoryRNA or recombinase for germ cell ablation can be the 5′ non-codingregion or a portion of the 5′ coding region which confersdevelopmentally regulated expression of an operably linked nucleic acidencoding a cytotoxic protein, inhibitory RNA or recombinase for germcell ablation of any developmentally regulated gene, including, but notlimited to, Vasa, c-kit, Dppa3, daz1, Dnd1, Fkbp6, Fragilis, Fragilis-2,GDF-3, Mov1011, Nanog, Nanos2, Nanos3, oct3/4, Prdm1, Prdm14, Tex13,Tiar and TNAP, as described herein.

Homologues and variants of developmentally regulated promoters may beused according to the present invention.

Promoter homologues and promoter variants can be included in a transgenefor germ cell ablation according to the present invention. The terms“promoter homologue” and “promoter variant” refer to a promoter whichhas substantially similar functional properties to confer the desiredtype of expression, such as developmentally regulated or ubiquitousexpression, on an operably linked nucleic acid compared to thosedisclosed herein. For example, a promoter homologue or variant hassubstantially similar functional properties to confer developmentallyregulated expression on an operably linked nucleic acid compared toVasa, c-kit, Dppa3, daz1, Dnd1, Fkbp6, Fragilis, Fragilis-2, GDF-3,Mov1011, Nanog, Nanos2, Nanos3, oct3/4, Prdm1, Prdm14, Tex13, Tiarand/or TNAP promoters.

One of skill in the art will recognize that one or more nucleic acidmutations can be introduced without altering the functional propertiesof a given promoter. Mutations can be introduced using standardmolecular biology techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis, to produce promoter variants. As used herein,the term “promoter variant” refers to either a naturally occurring or arecombinantly prepared variation of a reference promoter, such as Vasa,c-kit, Dppa3, daz1, Dnd1, Fkbp6, Fragilis, Fragilis-2, GDF-3, Mov1011,Nanog, Nanos2, Nanos3, oct3/4, Prdm1, Prdm14, Tex13, Tiar and/or TNAPpromoters.

Structurally, homologues and variants of developmentally regulatedand/or ubiquitous promoters have at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orgreater, nucleic acid sequence identity to the reference developmentallyregulated and/or ubiquitous promoter and include a site for binding ofRNA polymerase and, optionally, one or more binding sites fortranscription factors.

Homologues and variants of the mouse vasa promoter of SEQ ID NO:1 arecharacterized by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater,nucleic acid sequence identity to SEQ ID NO:1. Further homologues andvariants of the mouse vasa promoter of SEQ ID NO:1 and includetranscription factor binding sites having sequences of SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and are characterized by at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or greater, nucleic acid sequence identityto SEQ ID NO:1.

Homologues and variants of the c-kit promoter of SEQ ID NO:2 arecharacterized by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater,nucleic acid sequence identity to SEQ ID NO:2.

Homologues and variants of the oct3/4 promoter of SEQ ID NO:39 arecharacterized by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater,nucleic acid sequence identity to SEQ ID NO:39.

It is known in the art that promoters from other species are functional,e.g. the human VASA promoter is functional in the mouse. Homologues andhomologous promoters from other species can be identified usingbioinformatics tools known in the art such as the Mammalian promoterdatabase, and the CSHL Rat Promoter Database (RnPD) (Xuan et al., 2005,Genome Biol 6:R72 and Zhao et al., 2005, Nucl Acid Res 33:D103-107).Bioinformatic tools to identify promoter regions are known in the art,for example PromoSer (Halees et al. 2003, Nucl. Acids. Res. 2003 31:3554-3559).

These and other developmentally regulated promoters are obtained andused according to well-known methodology. For example, to obtain thepromoter for inclusion in a transgene, a Bacterial Artificial Chromosome(BAC) DNA clone for the desired gene can be isolated from a BAC libraryor obtained for example from the BACPAC Resources Center (BPRC) at theChildren's Hospital Oakland Research Institute in Oakland, Calif., USA.Methods for recombineering (recombination-mediated genetic engineering)are known in the art, such as the lambda red recombination techniquedescribed in Oginuma M et al. 2008, Mech Dev, 125(5-6):432-440; Datsenkoand Wanner, 2000, PNAS USA, 97(12):6640-6645 and Zhang Y, et al 2000,Nature Biotechnology 18, 1314-1317. Fragments of full-length promoterscan also be used as long as they have the required developmentallyregulated activity.

TABLE 1 List of developmentally regulated promoters Promoter AlternativeNames vasa mouse vasa homologue; Mvh, Ddx4, DDX4 c-kit Kit, CD117, SCFRdazl Dazh, DAZH Dnd1 Dead end homolog 1 Dppa3 Stella, Pgc7 Fkbp6 FK506binding protein 6, Fkbp36 Fragilis mil-1, Ifitm3, interferon-inducedtransmembrane protein 3 Fragilis-2 mil-2, Ifitm1, interferon-inducedtransmembrane protein 1 GDF-3 Growth differentiation factor 3 Mov10l1Mov10 like-1, a putative RNA helicase Nanog Nanos2 nanos homolog 2(Drosophila) Nanos3 nanos homolog 3 (Drosophila) oct3/4 oct-4 Prdm1Blimp-1 Prdm14 PR domain-containing protein 14, PFM11 Tex13testis-expressed gene 13 Tiar TIAL1 TNAP Alp1, alkaline phosphataseliver, bone, kidney

TABLE 2 List of more tightly developmentally regulated promotersPromoter Alternative Names vasa mouse vasa homologue; Mvh, Ddx4, DDX4Dnd1 Dead end homolog 1 Fkbp6 FK506 binding protein 6, Fkbp36 Fragilismil-1, Ifitm3, interferon-induced transmembrane protein 3 Fragilis-2mil-2, Ifitm1, interferon-induced transmembrane protein 1 GDF-3 Growthdifferentiation factor 3 Mov10l1 Mov10 like-1, a putative RNA helicaseNanos2 nanos homolog 2 (Drosophila) Nanos3 nanos homolog 3 (Drosophila)Prdm1 Blimp-1 Tex13 testis-expressed gene 13 Tiar TIAL1

TABLE 3 List of binding sequences for the vasa mouse promoterBinding site for Binding Position Transcription on mouse Factorchromosome Strand Binding Sequence Arnt chr13: + GTTCTCACGTGGCCTG SEQ ID113450683- NO: 3 113450698 USF-1:USF-2 chr13: + CTCACGTGGC SEQ ID113450686- NO: 4 113450695 USF I chr13: + GCACGTGC 113442541- 113442548Evi-1 chr13: − AGGCAAGGCAACATAA SEQ ID 113454164- NO: 5 113454179 c-Mycchr13: + TTGTTCTCACGTGGCCTGTG SEQ ID 113450681- NO: 6 113450700

TABLE 4 List of binding sequences for the Dnd1 promoter Binding siteBinding for Position Transcription on mouse Factor chromosome StrandBinding Sequence Cdc5 chr18: + GTGTTAACGTCTGAA SEQ ID 36945231- NO: 736945245 GCNF-2 chr18: − TTCCTGGTCAAGGTCAGA SEQ ID 36930976- NO: 836930993 AP-4 chr18: − CACAGCTGGG SEQ ID 36942377- NO: 9 36942386 MEF-2Achr18: − CTATAAACAGACCTCT SEQ ID 36941570- NO: 10 36941585 CUTLIchr18: + GTCATAGATAAGCTT SEQ ID 36933222- NO: 11 36933236 CUTLI chr18: +CACCGAGAAGTATGA SEQ ID 36945773- NO: 12 36945787 PPAR-alpha chr18: +CAGCACTGCCTCATAGATGA SEQ ID 36929910- NO: 13 36929929 YY1 chr18: +GGACCGCCATCTGCCGGGGA SEQ ID 36935914- NO: 14 36935933 YY1 chr18: +GATCTGCCATCCTGCCTGCC SEQ ID 36942745- NO: 15 36942764

TABLE 5 List of binding sequences for the Fkbp6 promoter Binding siteBinding for Position Transcription on mouse Factor chromosome StrandBinding Sequence STAT5A chr5: − TTCCCGGCA 135827748- 135827756 STAT5Achr5: − CAATTCCTGGAACTC SEQ ID 135839634- NO: 16 135839648 STAT5Achr5: + TTCCAGGAATTGCACCACCTGGTG SEQ ID 135839637- NO: 17 135839660STAT5A chr5: − TTCCTAGAA 135842246- 135842254 STAT5A chr5: −TTCCCAGTAGTGGCGACCCCAAGA SEQ ID 135843102- NO: 18 135843125 FOXO4 chr5:− CTGTTGTTCACCAG SEQ ID 135834794- NO: 19 135834807 HOXA9B chr5: −TGAGAGGGTTTCGG SEQ ID 135829441- NO: 20 135829454 RORalpha2 chr5: −GGAAGTGGGTCAC SEQ ID 135827701- NO: 21 135827713 Meis-la chr5: −TGAGAGGGTTTCGG SEQ ID 135829441- NO: 22 135829454 E47 chr5: −GAGTCCAGGTGTTGGG SEQ ID 135829296- NO: 23 135829311 E47 chr5: −TCCACCAGGTGGTGCA SEQ ID 135839647- NO: 24 135839662

TABLE 6 List of binding sequences for the MOV1OL1 promoter Binding siteBinding for Position Transcription on mouse Factor chromosome StrandBinding Sequence PPAR-gamma-1 chr15: + GACTGGGCAAAAGTTCA SEQ ID88810711- NO: 25 88810727 FOXD3 chr15: − TATTGTTTGTTT SEQ ID 88804989-NO: 26 88805000 E47 chr15: + CAAGGCCTCTGGCGTT SEQ ID 88810828- NO: 2788810843 Pbx1a chr15: − GTCGTCAATCATGCC SEQ ID 88816001- NO: 28 88816015Nkx5-1 chr15: + CAAGCGTGTG SEQ ID 88824333- NO: 29 88824342 Arntl chr15:− TGGGGAACGTGTTCCC SEQ ID 88806970- NO: 30 88806985 Arnt1 chr15: −GTTAGCACGTGAAGGA SEQ ID 88813173- NO: 31 88813188

TABLE 7 List of binding sequences for the Tex13 promoter BindingBinding site for Position Transcription on mouse Binding Factorchromosome Strand Sequence Bach-1 chrX: − GGTGAGTCAGC SEQ ID 137344530-NO: 32 137344540 NF-E2 chrX: + AGCTGACTCAC SEQ ID 137344529- NO: 33137344539 Oct-B1 chrX: − CTCATTTACATAC SEQ ID 137346298- NO: 34137346310

Host non-human embryos lacking functional endogenous germ cells areprovided wherein the germ cells have been ablated by geneticengineering, chemical ablation methods or physical ablation methods. Inthese non-human host embryos, donor stem cells are able to populate,expand and differentiate towards the germline, germ cells and gametespreferentially, such that the resulting live non-human animals havegametes completely or substantially derived from the donor non-humanstem cells.

The term “ablate” and grammatical equivalents encompasses inhibition,inactivation, killing, induction of apoptosis, inhibition ofdifferentiation, inhibition of function and/or inhibition ofproliferation of endogenous germ cells, thereby rendering the germ cellsincapable of generating gametes.

A host non-human embryo can be an embryo of any of various animals,including non-human mammals, such as non-human primates and rodents.According to embodiments of the present invention, the host non-humanembryo is a rodent embryo, particularly a mouse or rat embryo.

Host non-human embryos lacking functional germ cells are providedaccording to embodiments of the present invention which include a“deleter” transgene encoding a cytotoxic protein.

The term “deleter transgene” or “deleter gene” refers to a transgeneencoding a cytotoxic protein configured to express the cytotoxic proteinin endogenous germ cells thereby rendering the germ cells incapable ofgenerating gametes or to a transgene encoding an inhibitory RNAconfigured to express the inhibitory RNA in germ cells thereby renderingthe germ cells incapable of generating gametes. The term “deletertransgene” or “deleter gene” further refers to a transgene encoding aprotein which can interact with an exogenously added compound renderingit cytotoxic.

The term “cytotoxic protein” refers to a protein which ablatesendogenous germ cells, that is, causes inhibition, inactivation,killing, induction of apoptosis, inhibition of differentiation,inhibition of function and/or inhibition of proliferation of endogenousgerm cells, thereby rendering the germ cells incapable of generatinggametes.

Non-limiting examples of cytotoxic proteins include diphtheria toxin Afragment (DTA, SEQ ID NO:35), attenuated DTA, tox-176, diphtheria toxinreceptor, truncated, nonbinding derivative of Pseudomonas exotoxin A(PE-40), Pseudomonas exotoxin PE-38, herpes simplex virus 1 thymidinekinase (HSV-tk), truncated HSV-tk, delta-thymidine kinase (Δ-TK), ricin,Shiga toxin, a gene capable of inducing apoptosis or cell death, such ascaspase-7 (Casp7) and caspase-9 (Casp9).

As used herein, the term “cytotoxic protein variant” refers to either anaturally occurring or a recombinantly prepared variation of a referencecytotoxic protein.

Homologues and variants of cytotoxic proteins described herein arecharacterized by conserved functional properties compared to thecorresponding cytotoxic protein.

Thus, for example, homologues and variants of cytotoxic proteins retainthe ability to promote cytotoxicity when expressed in a mammalian hostcell. Functional characteristics of the putative homologue or variantcan be assayed, for example, transient transformation of germ cells invitro to detect cell death and/or induction of apoptosis. Assays forcytotoxic activity include, but are not limited to, transformation of ahost cell with an expression cassette encoding a putative cytotoxicprotein homologue or variant, followed by measurement of cell deathand/or inhibition of expression of germ cell and or gamete cell markersin the host cell, where increased cell death or decreased expression ofgerm cell and or gamete cell markers in the host cell, compared tocontrol host cells is indicative of conserved cytotoxic proteinfunctional properties of an homologue or variant. Assays for measurementof cell viability and analysis of expression of germ cell and gametemarkers in the host cell are well-known in the art.

The terms “diphtheria toxin A fragment” and “(DTA)” are usedinterchangeably herein to refer to the catalytic domain (C) ofdiphtheria toxin. As is well-known, diphtheria toxin is comprised of twopolypeptide fragments, A and B (Zdanovskaia, M. V. et al., Research inMicrobiology, 2000, 151, 557-562; Bennet, M. J. et al., Protein Science,1994, 3, 1444-1463). Fragment A (DTA) consists of the catalytic domain(C), whereas fragment B is made up of the receptor domain, (R), and thetransmembrane domain, (T). The R domain contains a receptor portionwhich binds to the BE-EGF receptor on the cell surface (Raab, G. et al.,Biochim. Biophys. Acta (BBA)/Reviews on Cancer 1997, 1333, F179-F199).The bound toxin then enters the cytoplasm by endocytosis. The C-terminushydrophobic series of α-sheets, known as the T domain, then embedsitself into the membrane, causing the N-terminus C domain to be cleavedand translocated into the cytoplasm. Once cleaved, the C domain becomesan active enzyme, catalyzing the creation of ADF-ribose-EF-2 from theprotein synthesis translocation peptide EF-2 and NAD+ (Hudson T H et al,J Biol. Chem. 1985 Mar. 10; 260(5):2675-80). Cytotoxic activity ofdiphtheria toxin, including DTA, homologues, fragments and variantsthereof is characterized by inhibition of protein synthesis of a cellcontaining the diphtheria toxin, including DTA, a homologue, a fragmentand/or a variant thereof.

Diphtheria toxin A fragment is set forth herein as SEQ ID NO:47 and anucleic acid sequence encoding Diphtheria toxin A fragment is set forthherein as SEQ ID NO:35.

Methods and compositions are not limited to DTA having the amino acidsequence of SEQ ID NO:47. Variants and fragments of DTA havingsubstantially similar cytotoxic activity may be used.

The term “diphtheria toxin A fragment” encompasses homologue and variantcytotoxic DTA proteins encoded by: 1) a nucleic acid sequence that hasat least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity tothe nucleic acid sequence set forth in SEQ ID NO:35 or a fragmentthereof; 2) the complement of a nucleic sequence that hybridizes underhigh stringency hybridization conditions to the nucleic acid set forthin SEQ ID NO:35, or a fragment thereof; an amino acid sequence that hasat least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity tothe amino acid sequence set forth in SEQ ID NO:48 or a cytotoxicfragment thereof.

As will be appreciated by one of skill in the art, due to the degeneracyof the genetic code, more than one nucleic acid will encode an identicalprotein. Thus, nucleic acids encoding DTA of SEQ ID NO:47 are notlimited to SEQ ID NO:35.

A fragment of DTA protein useful in the present invention is anyfragment of a DTA protein that is operable in the described methodsutilizing DTA.

A Diphtheria toxin A mutant, tox-176, is set forth herein as SEQ IDNO:48.

Methods and compositions are not limited to tox-176 protein having theamino acid sequence of SEQ ID NO:48. Variants and fragments of tox-176protein having substantially similar cytotoxic activity may be used.

The term “tox-176” encompasses homologue and variant cytotoxic tox-176proteins having an amino acid sequence that has at least 60%, 65%, 70%,72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequenceset forth in SEQ ID NO:48 or a cytotoxic fragment thereof.

A cytotoxic fragment of tox-176 protein useful in the present inventionis any fragment of a tox-176 protein that is operable in the describedmethods utilizing tox-176.

Herpes simplex virus thymidine kinase is set forth herein as SEQ IDNO:50 and a nucleic acid sequence encoding Herpes simplex virusthymidine kinase is set forth herein as SEQ ID NO:49.

Methods and compositions are not limited to Herpes simplex virusthymidine kinase having the amino acid sequence of SEQ ID NO:50.Variants, homologues, mutants and fragments of Herpes simplex virusthymidine kinase having similar cytotoxic activity may be used. Oneexample for a functional variant which retains thymidine kinase activityand can phosphorylate nucleoside analogs, such as ganciclovir, is Δ-TK(Salomon et al 1995 Mol Cell Bio, 15(10), 5322-5328).

The term “Herpes simplex virus thymidine kinase” encompasses homologueand variant Herpes simplex virus thymidine kinase proteins cytotoxic incombination with a thymidine analog and encoded by: 1) a nucleic acidsequence that has at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%,84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater identity to the nucleic acid sequence set forth in SEQ ID NO:49or a fragment thereof; 2) the complement of a nucleic sequence thathybridizes under high stringency hybridization conditions to the nucleicacid set forth in SEQ ID NO:49, or a fragment thereof; an amino acidsequence that has at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%,84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater identity to the amino acid sequence set forth in SEQ ID NO:50 ora fragment thereof cytotoxic in combination with a thymidine analog.

As will be appreciated by one of skill in the art, due to the degeneracyof the genetic code, more than one nucleic acid will encode an identicalprotein. Thus, nucleic acids encoding Herpes simplex virus thymidinekinase of SEQ ID NO:50 are not limited to SEQ ID NO:49.

A fragment of Herpes simplex virus thymidine kinase protein useful inthe present invention is any fragment of a Herpes simplex virusthymidine kinase protein that is operable in the described methodsutilizing Herpes simplex virus thymidine kinase such as a truncatedHSV-tk which retains thymidine kinase activity and can phosphorylatenucleoside analogs, such as ganciclovir.

The term “cytotoxic protein” also encompasses proteins which, incombination with an administered agent or compound cause inhibition,inactivation, killing, induction of apoptosis, inhibition ofdifferentiation, inhibition of function and/or inhibition ofproliferation of endogenous germ cells, thereby rendering the germ cellsincapable of generating gametes. For example, thymidine kinase is acytotoxic protein in the presence of an administered agent which is athymidine analog, such as ganciclovir (GCV) or a ganciclovir derivative,such as acyclovir or fialuridine.

The term “cytotoxic protein” encompasses homologues of cytotoxicproteins and variants thereof. The term “cytotoxic protein” furtherencompasses functionally active cytotoxic protein fragments.

According to embodiments of the present invention, expression of thedeleter transgene encoding a cytotoxic protein is developmentallyregulated, inducibly regulated or regulated by action of a site-specificrecombinase.

In particular embodiments the deleter transgene encoding a cytotoxicprotein is configured to express the cytotoxic protein in germ cells ofthe embryo using a developmentally regulated promoter operably linked toa nucleic acid encoding the cytotoxic protein. The developmentallyregulated promoter may confer germ cell-specific expression of thecytotoxic protein or may be active to express the cytotoxic protein innon-germ cells as well as in germ cells.

According to embodiments of the present invention, a transgenic hostembryo contains: a transgene encoding HSV-tk, a truncated HSV-tk or Δ-TKoperably linked to a developmentally regulated promoter enablingexpressing in germ cells. Administration of a thymidine analog, such asganciclovir (GCV) or a ganciclovir derivative, such as acyclovir orFialuridine (1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil,FIAU), induces depletion of cells expressing HSV-tk or a truncatedHSV-tk or Δ-TK, thereby ablating germ cells. The administration of thethymidine analog can be done orally or by subcutaneous (s.c.), i.p. orintravenously (i.v.) injection and is performed at least one time orrepeatedly. An example of a HSV-tk system is described in Zhang, Y. etal. 2005 FEBS T 272, 2207-15; Braun et al. 2000, Nature Medicine 6,320-326; Borrelli E., et al 1988, PNAS USA, 85 (20), 7572-7576; Chen etal. 2004, Nucl Acids Res 32 (20), e161, Salomon et al 1995 Mol Cell Bio,15(10), 5322-5328; Cohen et al 1998, Transgenic Res 7, 321-330.

Thus, according to embodiments, the pregnant female is treated with athymidine analog, such as GCV or FIAU to ablate germ cells expressingHSV-tk, a truncated HSV-tk or Δ-TK in the embryos at any embryo stagefrom the embryological stages E6 to E13, or a corresponding stage in anon-mouse species. According to embodiments, the pregnant female istreated with a thymidine analog, to ablate germ cells expressing HSV-tk,a truncated HSV-tk or Δ-TK in the embryos at an embryo stage fromembryological stage E6.5 to E12.5 or a corresponding stage in anon-mouse species. The thymidine analog will be administered, once, orseveral times daily over several consecutive days, e.g. on days E6.5,E7.5, E.8.5, E9.5, E10.5, E11.5 and E12.5; or intermittently, e.g. E6.5,E8.5, E10.5 or other intervals. For FIAU an amount from 10 to 50mg/kg/day will be administered. For GCV any amount from 20-100 mg/kg/daymay be administered.

Host non-human embryos lacking endogenous functional germ cells areprovided according to embodiments of the present invention which includea deleter transgene encoding a cytotoxic protein, the deleter transgeneconfigured to inducibly express the cytotoxic protein to ablate germcells in response to administration of an exogenous inducing agent tothe embryo, thereby rendering the germ cells incapable of generatinggametes.

Inducible promoter systems include, but are not limited to, the tet-offsystem, tet-on system, the Cre-ERT (Cre recombinase fused to a mutatedligand binding domain of the human estrogen receptor) or Cre-ERT2 (Crerecombinase fused to a G400V/M543A/L544A triple mutation of the humanestrogen receptor ligand binding domain also known as Crerecombinase-estrogen receptor T2) system (Metzger and Chambon 2001,Methods 24 (1), 71-80; Yamaguchi et al. 2009, Development 136,4011-4020; Monvoisin, et al., 2006, Dev Dyn 235, 3413-3422) and theHerpes simplex virus thymidine kinase (HSV-tk) system (Borrelli et al1988 PNAS USA, 85 (20), 7572-7576; Cohen et al 1998 Transgenic Res 7,321-330).

According to embodiments of the present invention, a transgenic hostembryo contains: a first transgene encoding a cytotoxic protein operablylinked to a Tet Operator sequence (TO) and a ubiquitous promoter or adevelopmentally regulated promoter; and a second transgene encoding aTet Repressor protein (TetR) operably linked to a second promoter whichis a ubiquitous promoter or a developmentally regulated promoter.Administration of a tetracycline or a tetracycline derivative, such asdoxycycline, induces expression of the cytotoxic protein, therebyablating germ cells. The Tet system is well known in the art, anddescribed in for example Wang J. et al. 2007 PNAS 104 (52): 20850-20855;Sheng et al. 2010, BMC Dev Biol, 10:17; Zhang et al., 2007, RNA 13(8),1375-1383.

According to embodiments of the present invention, a developmentallyregulated promoter operably linked to a nucleic acid encoding acytotoxic protein is further operably linked with an inducible promotersystem.

Host embryos lacking functional germ cells according to embodiments, arecreated by breeding one animal strain each carrying one of twotransgenes A and B with the following characteristics. Transgene Aexpresses a Tet Repressor Protein (TetR) driven by a ubiquitous promoteror a promoter active in germ cells and/or germ cells and/or germ cellderivatives and transgene B a Tet Operator sequence (TO) under thecontrol of a ubiquitous promoter or a promoter active in germ cellsand/or germ cell derivatives and operably linked to a nucleic acidencoding a cytotoxic protein and administering tetracycline or atetracycline derivative, such as doxycycline to induce expression of thecytotoxic protein.

Host mouse embryos lacking functional germ cells according toembodiments, are created by breeding one mouse strain each carrying oneof two transgenes A and B with the following characteristics. TransgeneA expresses a Tet Repressor Protein (TetR) driven by a ubiquitouspromoter or a promoter active in germ cells and/or germ cells and/orgerm cell derivatives and transgene B a Tet Operator sequence (TO) underthe control of a ubiquitous promoter or a promoter active in germ cellsand/or germ cell derivatives and operably linked to a nucleic acidencoding a cytotoxic protein and administering tetracycline or atetracycline derivative, such as doxycycline to induce expression of thecytotoxic protein.

Advantageously, the administration of the tetracycline or a tetracyclinederivative can be performed following transfer of embryos to apseudopregnant female tetracycline or a tetracycline derivative can beadministered in various ways, e.g. via the drinking water, in foodpellets, intraperitoneal injection (i.p.) or subcutaneous implantationof slow-release pellets (PNAS USA, Vol. 91, 9302-9306). Typically, donorstem cells are introduced into the embryo prior to administration of thetetracycline or a tetracycline derivative.

Thus, according to embodiments, the pregnant female is treated withtetracycline or a tetracycline derivative to induce expression of thecytotoxic protein or RNA interference molecule in the embryos at anyembryo stage from embryological stages E6 to E14, or a correspondingstage in a non-mouse species. According to embodiments, the pregnantfemale is treated with tetracycline or a tetracycline derivative toinduce expression of the cytotoxic protein in the embryos at any embryostage from embryological stages E6.5 to E10.5 or a corresponding stagein a non-mouse species.

According to embodiments of the present invention, a transgenicnon-human host embryo contains: a first transgene (the “deletertransgene”) encoding a cytotoxic protein operably linked to a firstpromoter and containing a cytotoxic protein inhibitory sequence operablylinked to recombinase recognition sites called “acceptor sequences;” anda second transgene (the “activator transgene”) encoding a site-specificrecombinase operably linked to a second promoter.

The “acceptor sequences” included in the deleter transgene allow precisedeletion of a nucleic acid positioned between the acceptor sequences byaction of the site-specific recombinase. Site-specific recombinases arewell-known in the art, exemplified by, but not limited to Cre, amodified Cre, Flp, Dre, Flpe, Flpo and phiC31. The acceptor sequencesare recombinase specific and are called loxP or variants thereof forCre; frt or variants thereof for Flp, Flpe and Flpo; rox or variantsthereof for Dre; attP/13 or variants thereof for phiC31.

Animals, such as mice or rats, expressing a site-specific recombinase,such as Cre recombinase, from a transgene under control of adevelopmentally regulated promoter as listed in Table 1 are generatedusing well-known methodology. Examples of existing mouse strainsexpressing a site-specific recombinase, such as Cre recombinase, from atransgene under control of a developmentally regulated promoter used inmethods and compositions according to embodiments of the presentinvention are listed in Table 8.

In FIG. 1 a schematic representation of a DNA construct containing asite specific recombinase gene to create an activator mouse strain isshown. GC Promoter refers to a tissue-specific or developmentallyregulated promoter suitable for developmentally regulated geneexpression in germ cells. Intron refers to an intron necessary forexpression, e.g. beta globin intron. Cre-p(A) refers to the geneencoding Cre recombinase with a N-terminal nuclear localization signaland 3′ p(A) signal. Sequence length are not drawn to scale. FIG. 2 showsa schematic representation of a DNA construct containing a site specificrecombinase gene to create an activator mouse strain using the vasapromoter. Vasa Promoter refers to the promoter region of vasa, e.g. a5.6 kb genomic fragment as described in Gallardo et al 2007, Genesis 45,413-417 and at least 80% identical to SEQ ID NO:1, suitable fordevelopmentally regulated Rene expression in germ cells. Intron refersto an intron necessary for expression, e.g. beta globin intron. Cre-p(A)refers to the gene encoding Cre recombinase with a N-terminal nuclearlocalization signal and 3′ p(A) signal. Sequence length are not drawn toscale.

TABLE 8 Cre expressing mouse strains Mouse Strain Name PromoterReference FVB-Tg(Ddx4-cre)1Dcas/J  Vasa (Ddx4, Gallardo, et al., 2007,Genesis mvh) 45, 413-417 B6.FVB-Tg(Ddx4- Vasa (Ddx4, Gallardo, et al.,2007, Genesis cre)1DCas/J mvh) 45, 413-417. FVB-Tg(Ddx4- cre)1Dcas/Jmice were backcrossed to the C57Bl/6J. Nanos3tm2.1(cre)Ysa Nanos-3Suzuki et al. 2007, Develop- ment 134, 77-83 Tg(Kit-cre)143Hmb c-kitBergqvist et al., FEBS Lett 438: 76 ± 80. 129-Alpl<tm1(cre)Nagy>/J Alp1Lomeli H et al 2000, Genesis (TNAP) 26: 116-117; The Jackson Laboratorystock no. 008569 Prdm1-Cre Prdm1 Ohinata Y et al 2005, Nature (Blimp1)436: 207-213 Dppa3-MCM Dppa3 Hirota T et al 2011, Biol Reproduction 85(2), 367-377

The first promoter, present in the deleter transgene and drivingexpression of the cytotoxic protein, can be a ubiquitous promoter or adevelopmentally regulated promoter (such as a germ cell-specificpromoter) and the second promoter, present in the activator transgeneand driving expression of the site-specific recombinase, is a ubiquitouspromoter or a developmentally regulated promoter (such as a germcell-specific promoter), wherein at least one of the promoters isdevelopmentally regulated.

An example is illustrated in FIG. 3 with a schematic representation of aDNA construct containing a toxic gene to create a deleter mouse strain.Homology arms (“ROSA 26”) to the mouse Gt(ROSA)26Sor locus are shownflanking the construct. The construct includes a splice acceptor site(SA), the reporter gene green fluorescent protein (GFP), a neomycinphosphotransferase coding sequence (Neo) with the PGK promoter andtranscription termination and mRNA polyadenylation (pA) signal, and bothflanked by loxP sites (LoxP), followed by a sequence that encodes for adiphtheria toxin fragment (DTA) followed by a transcription terminationand mRNA polyadenylation (pA) signal (also see Ivanova et al 2005,Genesis 43 (3), 129-135). Sequence lengths are not drawn to scale.

The deleter transgene inhibitory sequence is 1) one or more cytotoxicprotein-binding sequences which inhibit the activity of the cytotoxicprotein and/or 2) one or more stop codons which inhibit the expressionof the deleter transgene. A non-limiting example of a cytotoxic proteininhibitory sequence is a loxP reporter gene transcription terminationsignal loxP (floxed reporter stop).

In one embodiment, transgenic host non-human embryos are created bycrossing of two animal strains: 1) a first animal strain carrying anactivator transgene (“activator strain”) and 2) a second animal straincarrying a deleter transgene (“deleter strain”) generating transgenichost non-human embryos where germ cells are ablated and develops areceptive, empty germ cell niche.

In one embodiment, transgenic host non-human embryos are created bycrossing of two rat strains: 1) a first rat strain carrying an activatortransgene (“activator strain”) and 2) a second rat strain carrying adeleter transgene (“deleter strain”) generating transgenic host ratembryos where germ cells are ablated and develops a receptive, emptygerm cell niche.

In one embodiment, transgenic host non-human embryos are created bycrossing of two mouse strains: 1) a mouse strain carrying an activatortransgene (“activator mouse strain”) and 2) a mouse strain carrying adeleter transgene (“deleter mouse strain”) generating transgenic hostmouse embryos where germ cells are ablated, developing a receptive,empty germ cell niche.

An activator animal strain contains (a) a site-specific recombinase geneoperably linked to a germ cell specific or developmentally regulatedpromoter causing the expression of a site-specific recombinase gene ingerm cells.

According to embodiments of the present invention, the site-specificrecombinase is selected from the group consisting of Cre, a modifiedCre, Hp, Dre, Flpe, Flpo and phiC31. Optionally, the nucleic acidencoding the site-specific recombinase is operably linked to a nuclearlocalization sequence and/or intron sequences to enhance expression.

A deleter animal strain contains a deleter transgene encoding acytotoxic protein, and a cytotoxic protein inhibitory sequence disposedbetween a pair of acceptor sequences. The cytotoxic protein inhibitorysequence inhibits expression and/or cytotoxic effect of the cytotoxicprotein. For example, a cytotoxic inhibitory sequence preventstranscription of the deleter transgene such that the cytotoxic proteinis not expressed, such as one or more stop codons which inhibit theexpression of the deleter transgene. In a further example, a deletertransgene inhibitory sequence is one or more inhibitory RNA-bindingsequences which inhibit the activity of the inhibitory RNA. Anon-limiting example of an inhibitory sequence is a loxP reporter genetranscription termination signal loxP (foxed reporter stop).

Removal of the cytotoxic protein inhibitory sequence by the action ofthe site specific recombinase on the acceptor sites permits expressionand/or activity of the cytotoxic protein on the germ cells and theirablation.

Acceptor sequences corresponding to the site specific recombinase areincluded such as loxP, Frt, rax and attP/B.

Optionally, the deleter animal strain further encodes a reporter genethat allows detection or identification of cells expressing the reportergene.

According to embodiments of the present invention the site-specificrecombinase encoded by the activator transgene of the activator animalstrain is Cre and the acceptor sequences included in the deletertransgene of the deleter animal strain are loxP sequences.

According to embodiments of the present invention the site-specificrecombinase encoded by the transgene of the activator mouse is Cre, theacceptor sequences included in the transgene of the deleter mouse areloxP sequences the cytotoxic protein encoded by the transgene of thedeleter mouse is DTA, the first promoter is a Rosa26 promoter, and anucleotide sequence containing a DTA inhibitor disposed between theRosa26 promoter and the DTA gene is operably linked to at least two loxPsites. In the presence of Cre the foxed nucleotide sequence is removedand the Rosa26 promoter then drives the transcription of the DTA gene,ablating the germ cells.

In another embodiment, the deleter animal strain contains two acceptorsequences in an inverted orientation on one chromosome. In oneembodiment the acceptor sequences are loxP and the chromosome is mousechromosome number 2, when Cre is expressed an aberrant recombinationevent will occur. The resulting genomic rearrangements lead to celldeath.

In one embodiment both the loxP sites are in the HoxD complex of mousechromosome number 2.

In one embodiment host preimplantation embryos are generated by crossinghomozygous ROSA26-DTA176 female mice (Wu et al. 2006, Development133:581-90) with a mouse strain homozygous expressing the Crerecombinase under a developmentally controlled promoter as listed inTable 2. The F1 offspring will have a foxed DTA gene resulting in all F1embryos eliminating their own germ cells during development and fail todevelop sperm.

In a further embodiment host preimplantation embryos are generated bycrossing homozygous ROSA26-DTA176 female mice with homozygousB6.FVB-Tg(Ddx4-cre)1DCas/J (derived from by Gallardo et al Genesis45(6), 413-7 t C57BL/6J) male mice. After crossing all derived F1embryos carry both the Cre recombinase under a Vasa promoter control andthe foxed DTA gene. This results in all F1 embryos eliminating their owngerm cells during development. Preimplantation embryos are isolated andstem cells are introduced by methods known in the art. The resultingchimeras are used in breeding to generate stem-cell derived offspring.

Host non-human embryos lacking endogenous functional germ cells areprovided according to embodiments of the present invention which includea “deleter” transgene encoding an inhibitory RNA capable of silencing ordisrupting gene expression of one or more genes required for normaldevelopment of endogenous germ cells in a non-human animal.

The term “inhibitory RNA” refers to RNA molecules active to specificallydecrease levels or function of a target RNA in endogenous germ cellsthereby rendering the germ cells incapable of generating gametes.Inhibitory RNA includes antisense RNA, RNAi, shRNA, shRNA and micro RNA(miRNA).

RNA interference is a target sequence-specific method of inhibiting aselected gene. RNA interference has been characterized in numerousorganisms and is known to be mediated by a double-stranded RNA, alsotermed herein a double-stranded RNA compound. Briefly described, RNAinterference involves a mechanism triggered by the presence of smallinterfering RNA, siRNA, resulting in degradation of a targetcomplementary mRNA. siRNA is double-stranded RNA which includes anucleic acid sequence complementary to a target sequence in the gene tobe silenced. The double-stranded RNA may be provided as a longdouble-stranded RNA compound, in which case it is subject to cleavage bythe endogenous endonuclease Dicer in a cell. Cleavage by Dicer resultsin siRNA duplexes having about 21-23 complementary nucleotides in eachof the sense strand and the antisense strand, and optionally 1-2nucleotide 3′ overhangs on each of the two strands.

Alternatively, siRNA is provided as a duplex nucleic acid having a sensestrand and an antisense strand, wherein the sense and antisense strandsare substantially complementary and each of the sense and antisensestrands have about 16-30 nucleotides. The complementary sense andantisense strands and optionally include 1-2 nucleotide 3′ overhangs onone or both of the two strands. In one embodiment, a siRNA is preferredwhich has sense and antisense strands, wherein each of the two strandshas 21-23 nucleotides, wherein 2 nucleotides on the 3′ end of eachstrand are overhanging and the remaining 19-21 nucleotides are 100%complementary. As noted above, further details of siRNA compounds aredescribed in Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts ofRNAi Technology, DNA Press LLC, Eagleville, Pa., 2003. Additionaldescription of siRNA length and composition is found in Elbashir, S. M.et al., Gene Dev 15:188-200, 2001; and O'Toole, A. S. et al., RNA,11:512-516, 2005.

siRNA provided as a duplex nucleic acid having a sense strand and anantisense strand may be configured such that the sense strand andantisense strand form a duplex in hybridization conditions but areotherwise unconnected. A double-stranded siRNA compound may be assembledfrom separate antisense and sense strands. Thus, for example,complementary sense and antisense strands are chemically synthesized andsubsequently annealed by hybridization to produce a syntheticdouble-stranded siRNA compound.

Further, the sense and antisense strands for inclusion in siRNA may beproduced from one or more expression cassettes encoding the sense andantisense strands. Where the sense and antisense strands are encoded bya single expression cassette, they may be excised from a producedtranscript to produce separated sense and antisense strands and thenhybridized to form a duplex siRNA. See, for example, Engelke, D. R., RNAInterference (RNAi): Nuts and Bolts of RNAi Technology, particularlychapters 5 and 6, DNA Press LLC, Eagleville, Pa., 2003 for furtherdetails of synthetic and recombinant methods of producing siRNA.

In a further alternative, a double-stranded “short hairpin” RNAcompound, termed “shRNA” or “hairpin siRNA” includes an antisense strandand a sense strand connected by a linker. shRNA may be chemicallysynthesized or formed by transcription of a single-stranded RNA from anexpression cassette in a recombinant nucleic acid construct. The shRNAhas complementary regions which form a duplex under hybridizationconditions, forming a “hairpin” conformation wherein the complementarysense and antisense strands are linked, such as by a nucleotide sequenceof about 1-20 nucleotides. In general, each of the complementary senseand antisense strands have about 16-30 nucleotides.

As noted, siRNA and shRNA may be expressed from a DNA template encodingthe desired transcript or transcripts. A DNA template encoding thedesired transcript or transcripts is inserted in a vector, such as aplasmid or viral vector, and operably linked to a promoter forexpression in vitro or in vivo.

As will be recognized by one of skill in the art, particular siRNAs maybe of different size and still be effective to inhibit a target gene.Routine assay may be performed to determine effective size andcomposition of particular compounds. Without wishing to be bound bytheory, it is believed that at least the antisense strand isincorporated into an endonuclease complex which cleaves the target mRNAcomplementary to the antisense strand of the siRNA.

Administration of long RNA duplexes processed to siRNA, as well asadministration of siRNA or shRNA, and/or expression constructs encodingsiRNA or shRNA, results in degradation of the target mRNA and inhibitionof expression of the protein encoded by the target mRNA, therebyinhibiting activity of the encoded protein in the cell.

Further details of RNA interference mechanisms as well as descriptionsof target identification, synthetic siRNA and shRNA production, siRNAand shRNA expression construct production, and protocols forpurification and delivery of expression constructs and synthetic siRNAand shRNA in vitro and in vivo are described in Engelke, D. R., RNAInterference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC,Eagleville, Pa., 2003.

Inhibitory RNA used in the present invention is capable of silencing ordisrupting gene expression of one or more genes required for normaldevelopment of germ cells in a non-human animal.

Examples of such genes include, but are not limited to, those listed inTable 9.

TABLE 9 Gene Reference for cDNA vasa (Mvh, Ddx4) Fujiwara et al., 1994,Proc. Natl. Acad. Sci. USA 91, 12258-12262 and GenBank no. G65195 c-kitGeissler et al., 1988, Cell, 55, 185- 192 Qiu et al., 1988, EMBO J.7(4): 1003-1011. GenBank Y00864.1 dazl (also known as dazh or Reijo etal., 1996, Genomics 35: 346- DAZH) 352 and GenBank no. NM_010021 Dnd1(dead end homolog 1) Youngren et al. 2005, Nature 435, 360- 364;Bhattacharya C et al. Biochem Biophys Res Commun. 2007, 355(1): 194-9.Dppa3 (also known as stella Saitou et al., 2002, Nature 418, or Pgc7)stella (PGC7, 293-300 Dppa3) Fkbp6 (FK506 binding protein Crackower etal., 2003, Science 300, 6 or Fkbp36)Fkbp6 1291-1295 Mov10l1(Mov10-like-1) Wang et al., 2001, Nat Genet 27: 422- 426 and GenBank no.AF285587 nanog Wang et al., 2003, Gene Expr. Patterns 3 (1), 99-103 andGenBank no. AF507043 nanos2 Tsuda M et al., 2003, Science 301, 1239-1241nanos3 Tsuda M et al., 2003, Science 301, 1239-1241 oct3/4 (also knownas oct-4, Okazawa et al 1991, EMBO J. 10 (10), Pou5f1 or POU domain,class 2997-3005; Mizuno and Kosaka 2008, 5, transcription factor 1) J.Biol. Chem. 283 (45), 30997-31004 and GenBank no. S58422 Prdm1 (Blimp-1)Turner et al., 1994, Cell 77, 297-306. Prdm14 (PR domain-contain- YamajiM et al. 2008, Nature Genetics ing protein 14, also known 40, 1016-1022as PFM11) sox2 Collignon et al., 1996, Development 122 (2), 509-520 andGenBank no. X94127 tex13 (Testis expressed gene Wang et al., 2001, NatGenet 27: 422- 13) 426 and GenBank no. AF285576; Tiar (Tial1) Beck etal., 1996, Nucleic Acids Res. 24, 3829-3835 TNAP (Alpl, alkalineMacGregor G R et al (1995) De- phophatase liver, bone, kidney)velopment, 121 (5), 1487-1496.

Inhibitory RNAs directed to specific genes are commercially availablefrom many suppliers such as OriGene, Life Technologies (Invitrogen),Santa Cruz Biotechnology, Sigma-Aldrich and others. Bioinformatic toolsknown in the art can be used to design the RNA interference molecule fora specific target gene, such as Gene Link, the RNAi designer fromClontech etc.

FIG. 4 shows a schematic representation of a DNA construct containing aU6 promoter (U6) followed by a loxP (LoxP) site, followed by the widelyexpressed CMV promoter, the reporter gene green fluorescent protein(GFP) and the second loxP site, then followed by the shRNA or RNAispecific for a developmentally regulated gene such as oct3/4 and atranscription termination and mRNA polyadenylation (pA) signal. Afterthe Cre mediated recombination GFP is flipped out and the shRNAexpression is driven by the U6 promoter. Sequence lengths are not drawnto scale.

Examples of target RNA for shRNA are vasa, Prdm14 for example asdescribed in Chia et al., 2010, Nature 468, 316-32; Nanos2, Nanos3,oct3/4 (also known as Pou5f1 or POU domain, class 5, transcriptionfactor 1) for example as described in Ivanova N et al., 2006, Nature442, 533-538, Blimp-1 (also known as Prdm1), as described in Turner etal., 1994, Cell 77, 297-306; stella (also known as PGC7 or Dppa3),c-kit, for example as described in Sikarwar and Reddy, Oligonucleotides2008, 18(2):145-60, daz1 for example as described in Yu et al., 2009, JMol Cell Biol, 1 (2): 93-103 and Ivanova N et al., 2006, Nature 442,533-538; Tiar, as described in Izquierdo, 2006 Biochem Biophys ResCommun 348, 2, 703-711, Dnd1; Fkbp6, Mov1011, nanog, for example asdescribed in Yamaguchi et al., Development 136, 4011-4020 (2009) andIvanova N et al., 2006, Nature 442, 533-538; sox2, for example asdescribed in Ivanova N et al., 2006, Nature 442, 533-538, and tex13.

In particular embodiments the deleter transgene encoding an inhibitoryRNA is configured to express the inhibitory RNA in germ cells of theembryo using a developmentally regulated promoter operably linked to anucleic acid encoding the inhibitory RNA. The developmentally regulatedpromoter may confer germ cell-specific expression of the inhibitory RNAor may be active to express the inhibitory RNA in non-germ cells as wellas in germ cells. A list of such promoters is provided in Table 1.

According to embodiments, developmentally regulated promoters are activein primordial germ cells and/or during germ cell development and/ordifferentiation. Promoters active in the early stages of germ celldevelopment are preferred in order to create a niche in the host embryoto reduce the competition of the donor stem cells with host cells.

Host non-human embryos lacking endogenous germ cells are providedaccording to embodiments of the present invention which include atransgene encoding an inhibitory RNA, the transgene configured toinducibly express the inhibitory RNA to ablate endogenous germ cells inresponse to administration of an exogenous inducing agent to the embryo,thereby rendering the germ cells incapable of generating gametes.

Inducible promoter systems include, but are not limited to, the tet-offsystem, tet-on system, the Cre-ERT (Cre recombinase fused to a mutatedligand binding domain of the human estrogen receptor) or Cre-ERT2 (Crerecombinase fused to a G400V/M543A/L544A triple mutation of the humanestrogen receptor ligand binding domain also known as Crerecombinase-estrogen receptor T2) system.

According to embodiments of the present invention, a transgenic hostembryo contains: a first transgene encoding an inhibitory RNA operablylinked to a Tet Operator sequence (TO) and a ubiquitous promoter or adevelopmentally regulated promoter as listed in Table 1; and a secondtransgene encoding a Tet Repressor protein (TetR) operably linked to asecond promoter which is a ubiquitous promoter or a developmentallyregulated promoter. Administration of a tetracycline or a tetracyclinederivative, such as doxycycline, induces expression of the inhibitoryRNA, thereby ablating germ cells. This is illustrated in FIGS. 5 and 6.FIG. 5 is a schematic representation of a DNA construct containing theCAG promoter, which is a combination of the cytomegalovirus (CMV) earlyenhancer element and modified chicken beta-actin promoter and intron 1followed by sequences encoding for the TetR protein and a transcriptiontermination and mRNA polyadenylation (pA) signal. Sequence length arenot drawn to scale. FIG. 6 is a schematic representation of a DNAconstruct containing a human RNAse P RNA H1 promoter (H1 promoter), atet operator (TO) followed by the short hairpin RNA or RNAi sequence anda transcription termination and mRNA polyadenylation (pA) signal.Sequence lengths are not drawn to scale.

According to embodiments of the present invention, a developmentallyregulated promoter operably linked to a nucleic acid encoding aninhibitory RNA is further operably linked with an inducible promotersystem.

Host mouse embryos lacking functional germ cells according toembodiments, are created by breeding one mouse strain each carrying oneof two transgenes A and B with the following characteristics. TransgeneA expresses a Tet Repressor Protein (TetR) driven by a ubiquitouspromoter or a promoter active in germ cells and/or germ cells and/orgerm cell derivatives and transgene B a Tet Operator sequence (TO) underthe control of a ubiquitous promoter or a promoter active in germ cellsand/or germ cell derivatives and operably linked to a nucleic acidencoding an inhibitory RNA and administering tetracycline or atetracycline derivative, such as doxycycline to induce expression of theinhibitory RNA.

Advantageously, the administration of the tetracycline or a tetracyclinederivative can be performed following transfer of embryos to apseudopregnant female mouse. Typically, donor stem cells are introducedinto the embryo prior to administration of the tetracycline or atetracycline derivative.

Thus, according to embodiments, the pregnant female is treated withtetracycline or a tetracycline derivative to induce expression of theinhibitory RNA in the embryos at an embryo stage from any of theembryological stages E6, E6.5, E7, E7.5 E8, E8.5, E9, E9.5, E10, E10.5,E11, E11.5, E12, E12.5, E13, E13.5 and E14, or corresponding stages in anon-mouse species. According to embodiments, the pregnant female istreated with tetracycline or a tetracycline derivative inducingexpression of the inhibitory RNA in the embryos at an embryo stage fromany embryological stage in the range of E6.5 to E10.5, inclusive, or acorresponding stage in a non-mouse species.

According to embodiments of the present invention, a transgenicnon-human host embryo contains: a first transgene (the “deletertransgene”) encoding an inhibitory RNA operably linked to a firstpromoter and containing an inhibitory sequence operably linked torecombinase recognition sites called “acceptor sequences;” and a secondtransgene (the “activator transgene”) encoding a site-specificrecombinase operably linked to a second promoter.

The “acceptor sequences” included in the deleter transgene allow precisedeletion of a nucleic acid positioned between the acceptor sequences byaction of the site-specific recombinase. Site-specific recombinases arewell-known in the art, exemplified by, but not limited to, Cre, amodified Cre, Flp, Dre, Flpe, Flpo and phiC31. The acceptor sequencesare recombinase specific and are called loxP or variants thereof forCre; frt or variants thereof for Flp, Flpe and Flpo; rox or variantsthereof for Dre; attP/B or variants thereof for phiC31. See Table 7 forexamples of Cre-expressing mouse strains.

The first promoter, present in the deleter transgene and drivingexpression of the inhibitory RNA, can be a ubiquitous promoter or adevelopmentally regulated promoter (such as a germ cell-specificpromoter) and the second promoter, present in the activator transgeneand driving expression of the site-specific recombinase, is a ubiquitouspromoter or a developmentally regulated promoter (such as a germcell-specific promoter), wherein at least one of the promoters isdevelopmentally regulated. A list of suitable developmentally regulatedpromoters is provided in Table 1.

The deleter transgene inhibitory sequence is 1) one or more inhibitoryRNA-binding sequences which inhibit the activity of the inhibitory RNAand/or 2) one or more stop codons which inhibit the expression of thedeleter transgene. A non-limiting example of an inhibitory sequence is aloxP reporter gene transcription termination signal loxP (foxed reporterstop).

In one embodiment, transgenic host non-human embryos are created bycrossing of two different animal strains: 1) a first animal straincarrying an activator transgene (“activator strain”) and 2) a secondanimal strain carrying a deleter transgene (“deleter strain”) generatingtransgenic host non-human embryos which develop a receptive, open germcell niche.

In one embodiment, transgenic host non-human embryos are created bycrossing of two different rat strains: 1) a first rat strain carrying anactivator transgene (“activator strain”) and 2) a second rat straincarrying a deleter transgene (“deleter strain”) generating transgenichost rat embryos which develop a receptive, open germ cell niche.

In one embodiment, transgenic host non-human embryos are created bycrossing of two different mouse strains: 1) a mouse strain carrying anactivator transgene (“activator mouse strain”) and 2) a mouse straincarrying a deleter transgene (“deleter mouse strain”) generatingtransgenic host mouse embryos which develop a receptive, open germ cellniche.

An activator animal strain contains (a) a site-specific recombinase geneoperably linked to a germ cell specific or developmentally regulatedpromoter causing the expression of a site-specific recombinase gene ingerm cells.

According to embodiments of the present invention, the site-specificrecombinase is selected from the group consisting of Cre, a modifiedCre, Flp, Dre, Flpe, Flpo and phiC31. Optionally, the nucleic acidencoding the site-specific recombinase is operably linked to a nuclearlocalization sequence and/or intron sequences to enhance expression.

A deleter animal strain contains a deleter transgene encoding acytotoxic protein, and a cytotoxic protein inhibitory sequence disposedbetween a pair of acceptor sequences. The cytotoxic protein inhibitorysequence inhibits expression and/or cytotoxic effect of the cytotoxicprotein. Removal of the cytotoxic protein inhibitory sequence by theaction of the site specific recombinase on the acceptor sites permitsexpression and/or activity of the cytotoxic protein in the germ cells.

Acceptor sequences corresponding to the site specific recombinase areincluded such as loxP, Frt, rox and attP/B.

Optionally, the deleter animal strain further encodes a reporter genethat allows detection or identification of cells expressing the reportergene.

According to embodiments of the present invention the site-specificrecombinase encoded by the activator transgene of the activator animalstrain is Cre and the acceptor sequences included in the deletertransgene of the deleter animal strain are loxP sequences.

According to embodiments of the present invention, a transgenic hostembryo contains: a first transgene encoding a oct3/4 RNA interferencemolecule operably linked to a H1-RNA Polymerase-III (pol III) promoteror the U6 promoter and containing a RNA interference molecule inhibitorysequence flanked on both sides by loxP sites and a second transgeneencoding Cre operably linked to a second promoter. In the presence ofCre, the floxed deleter gene inhibitory sequence is removed allowing theH1-RNA Polymerase-H1 or U6 promoter to drive the transcription of theoct3/4 RNA interference molecule, resulting in inhibition of oct3/4translation and arrested development of germ cells in the transgenichost embryo. A suitable oct3/4 target sequence is: GGATGTGGTTCGAGTATGGT(SEQ ID NO:36).

According to embodiments of the present invention, the reporter gene isoperably linked with a ubiquitous promoter (e.g. CMV or CAG or ROSA26)and operably linked to acceptor sequences (e.g. loxP or frt), 5′ of thefirst loxP is a promoter suitable for expression of shRNAs or siRNAssuch as the H1-RNA Polymerase-III or U6 promoter and 3′ of the secondloxP is the shRNA. The reporter gene is ubiquitously expressed in allcells carrying this construct. After the site-specific recombinationevent, the GFP and its promoter are removed and the shRNA or siRNA isexpressed and can inhibit expression of its target gene. In FIG. 4 aschematic representation of a DNA construct is shown. The constructcontains a U6 promoter (U6) followed by a loxP (LoxP) site, followed bythe widely expressed CMV promoter, the reporter gene green fluorescentprotein (GFP) and the second loxP site, then followed by the shRNA orRNAi specific for a developmentally regulated gene such as oct3/4 and atranscription termination and mRNA polyadenylation (pA) signal. Afterthe Cre mediated recombination GFP is flipped out and the shRNAexpression will be driven by the U6 promoter. Sequence lengths are notdrawn to scale.

In another embodiment, the deleter mouse strain contains two acceptorsequences in an inverted orientation on one chromosome. In a specificembodiment the acceptor sequences are loxP and the chromosome is mousechromosome number 2. In the presence of Cre recombinase cells containingthe inverted loxP will undergo genomic rearrangements resulting in celldeath. An example for the inverted loxP mouse strain is described inKmita et al., 2000, Nat Genet. 26:451-454.

An example of an inverted loxP construct is shown in FIG. 11. FIG. 10shows that F1 animals derived from breeding mice carrying an invertedloxP with a Vasa-Cre mouse have significantly smaller testes (right)when compared to wild type mice (left). Further these males areinfertile and the testes do not contain any sperm.

Ubiquitous promoters include, but are not limited to, CAG(cytomegalovirus early enhancer element and chicken beta-actin promoterand intron 1), CMV (cytomegalovirus), ROSA26, PGK-1 (3-phosphoglyceratekinase), beta-actin, heat shock protein 70 (Hsp70), EF-1 alpha geneencoding elongation factor 1 alpha (EF1α), eukaryotic initiation factor4A (eIF-4A1), H1-RNA Polymerase-III, and U6 promoter.

The host non-human embryos of the present invention are generated byvarious methods such as by genetic engineering, chemical modification orphysical modification of an embryo.

Expression constructs are provided according to embodiments of thepresent invention for generation of transgenic host non-human embryos.

Generation of a transgenic non-human animal described herein can beachieved by methods such as DNA injection of an expression constructinto a preimplantation embryo or by implantation of stem cellscontaining the expression construct, such as embryonic stem (ES) cellsor induced pluripotent stem (iPS) cells.

The generation of transgenic rats is well-known in the art asexemplified in Mullins et al. 1990, Nature, 344:541-544, Tesson et al.2005, Transgenic Res 14 (5), 531-546; Charreau et al., Transgenic Res 5(4), 223-234; and Tenenhaus Dann 2007, Transgenic Res, 16 (5), 571-580.Generation of transgenic cells and organisms including mouse and rats isoptionally accomplished using DNA injection, lentivirus injection, zincfinger nuclease or similar technologies (engineered zinc-fingernucleases) as described in Geurts et al, 2009, Science, 325 (5939), 433;Kawamata et al., 2010, PNAS USA 10, 14223-14228; Meyer et al. 2010, PNASUSA 24, 15022-15026 or by TALEN or TAL effector nuclease technology asdescribed in Bogdanove and Voytas, Science 2011, 333, 1843-1846 andScholze and Boch, Current Opinion in Microbiology 2011, 14:47-53.

According to embodiments of the present invention, expression constructsare provided which include a nucleic acid sequence encoding a deletergene operably linked to a germ cell-specific or developmentalstage-specific promoter.

In one embodiment, transgenic host non-human embryos are created bycrossing of two different rodent strains: 1) a rodent strain carrying anactivator (“activator rodent strain”) and 2) a rodent strain carrying adeleter transgene (“deleter rodent strain”) generating transgenic hostnon-human embryos which develop a receptive germ cell niche.

According to embodiments of the present invention, methods for making achimeric rodent are provided which include introducing at least onerodent donor stem cell into a rodent host embryo, wherein the hostembryo includes (i) a transgene encoding a site-specific recombinasegene operably linked to a developmentally-regulated promoter wherein thesite-specific recombinase gene is expressed in germ cells during anembryo stage, and (ii) a transgene encoding a deleter gene whoseexpression results in ablation of germ cells, wherein expression of thetransgene encoding the cytotoxic protein or RNA interference molecule isinduced by the enzymatic action of the site-specific recombinase. Thetransgenic host embryo including at least one donor stem cell isintroduced into a pseudopregnant female rodent where it is gestated toproduce live-born chimeric rodents wherein germ cells and gametesproduced by the chimeric rodent are derived from donor stem cellsintroduced into the transgenic host embryo.

According to embodiments of the present invention the rodent is a mouse.

According to embodiments of the present invention the rodent is a rat.

Non-human host embryos lacking germ cells can be cryopreserved forstorage and later use. For example, non-human host embryos lacking, ordestined to lack germ cells can be cryopreserved in a media comprising acryoprotectant. In a further example, non-human host embryos lackinggerm cells can be cryopreserved in a container at subzero temperatures.

Non-human host embryos lacking germ cells of the present invention haveutility to generate chimeric animals. One or more stem cells isintroduced into the non-human host embryo destined to lack functionalgerm cells, the embryo with the introduced stem cells is then gestatedunder suitable conditions, such as by introduction into a pseudopregnantfemale animal, producing a chimeric animal having all or substantiallyall germ cells and gametes derived from the stem cells.

The terms “stem cell” and “stem cells” are used interchangeably andrefer to pluripotent stem cells capable of differentiating into germcells, such as embryonic stem (ES) cells, epiblast stem cells (EpiSCs orepi stem cell), embryonic germ (E6) cells and induced pluripotent stem(iPS) cells. The term “stem cell” includes genetically modified stemcells.

Stem cells are cultured in conditions suitable for the particular stemcell line using methods known in the art, for example see Tremml et al.,2008, Current Protocols in Stem Cell Biology, Chapter 1:Unit 1C.4; BaehrM et al. 2003, Philosophical Transactions of the Royal Society B:Biological Sciences 358, 1397-140 and K. Turksen (Ed.) 2002.

A stem cell introduced into a transgenic host embryo can be a stem cellfrom any of various animals, including mammals, such as humans,non-human primates and rodents. According to embodiments of the presentinvention, the stem cell is a marmoset stem cell. According toembodiments of the present invention, the stem cell is a rodent stemcell. According to embodiments of the present invention, the stem cellis a mouse stem cell. According to embodiments of the present invention,the stem cell is a rat stem cell.

According to the embodiment of the present invention donor stem cellsare from the same species as the host embryo.

According to the embodiment of the present invention donor stem cellsare from a different mammalian species than the host embryo.

According to embodiments of the present invention, the stem cell isgenetically modified.

According to embodiments of the present invention, the stem cell is arat stem cell. Germline competent rat ES cells are well-known in the artas exemplified in Buehr et al., 2008, Biol. Reprod. 68: 222-229; Li etal., 2008, Cell 135: 1299-1310; Zhao et al., 2010, J. Genet. Genomics37, 467-473) and rat iPS cells in 2009 (Li et al., 2009, Cell Stem Cell4: 16-19; Liao et al., 2009, Cell Stern Cell 4:11-15. To generatechimeric rats, rat ES or iPS cells are injected into preimplantationembryos, such as blastocysts, using well-known methodology such asdescribed in Zhao et al., 2010, J. Genet. Genomics 37, 467-473; Popovaet al., 2005, Transgenic Res., 14(5):729-38).

According to embodiments of the present invention, the stem cell is amarmoset monkey (Callithrix jacchus) stem cell (e.g. Muller et al 2009,Hum. Reprod 24 (6): 1359-1372).

Methods according to embodiments of the present invention includeintroducing a donor stem cell into a non-human transgenic host embryo,growing the host embryo under suitable conditions to produce livechimeric animals wherein the gametes of the chimeric animals aresubstantially or wholly derived from the donor stem cells.

According to embodiments, methods are provided using any combination ofthe compositions and/or methods described herein to make a chimericmouse including introducing one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteencells into a mouse host preimplantation embryo that is a 2-cell stage, a4-cell stage, a 8-cell stage, a 16-cell stage, a 32-cell stage, a64-cell stage embryo, a morula or a blastocyst; and introducing thepreimplantation embryo including the stem cells into a mouse that iscapable of gestating the embryo. In one embodiment the hostpreimplantation embryo is a blastocyst and the number of donor stemcells is six to twelve cells. In one embodiment, the host embryo is an8-cell stage embryo and the number of donor stem cells is two to tencells.

Methods of generating chimeric animals having germ cells and/or gametessubstantially or wholly derived from donor stem cells are providedwherein the donor stem cells are introduced into a host preimplantationembryo of the present invention.

According to embodiments, stem cells are injected into a 2-cell, 4-cell,8-cell, 2-cell stage, a 4-cell stage, an 8-cell stage, a 16-cell stage,a 32-cell stage, a 64-cell, a morula and a blastocyst stage hostpreimplantation embryo of the present invention. The host embryos areselected from a pre-morula stage, a morula stage, an uncompacted morulastage, a compacted morula stage and a blastocyst stage. In oneembodiment the host embryos are selected from the embryological agestages E1, E1.5, E2, E2.5, E3 and E3.5 for mouse embryos. According toembodiments, stem cells are injected into a host embryo having adevelopmental stage selected from a Theiler Stage 2 (TS2), a TS3, a TS4,a TS5 and a TS6, with reference to the Theiler stages as described inTheiler (1989) The House Mouse: Atlas of Mouse Development, by TheilerSpringer-Verlag, NY.

In one embodiment the host embryo is selected from the Theiler stagesTS3, TS4 and TS5.

In a specific embodiment, the host embryo is a morula.

In a specific embodiment, the host embryo is a blastocyst.

Preimplantation embryos are isolated for introduction of stem cells.

For example, groups of single donor stem cells are selected using afinely drawn-out glass needle (20-25 micrometer inside diameter) andintroduced through the embryo's zona pellucida for early embryos andinto the blastocysts cavity (blastocoel) using an inverted microscopefitted with micro-manipulators for blastocysts. Approximately 9-10 stemcells (ES or iPS or epi stem cells) are injected per blastocysts, or8-cell stage embryo, 6-9 stem cells per 4-cell stage embryo, and about 6stem cells per 2-cell stage embryo. Stem cell injection may be assistedwith a laser or piezo pulses drilled opening the zona pellucida. (seeKraus et al. 2010, Genesis 48, 394-399). Alternatively, stem cells canbe aggregated with morula or injected into early stage embryos (e.g.2-cell, 4-cell, 8-cell, premorula or morula) with or without the zonapellucida.

Gestating the embryos under conditions suitable for development of theembryos is performed according to standard methodology. The non-humanembryos including donor stem cells are implanted into pseudopregnantfemales as known in the art (see Manipulating the Mouse Embryo: ALaboratory Manual, 3^(rd) edition (A. Nagy et al. 2002, CSHL Press,ISBN-10: 0879695919; Nagy et al., 1990, Development 110, 815-821; U.S.Pat. No. 7,576,259, U.S. Pat. No. 7,659,442, U.S. Pat. No. 7,294,754,Kraus et al. 2010, Genesis 48, 394-399). Briefly, fertile female rodentsbetween 6-8 weeks of age are mated with vasectomized or sterile rodentmales to induce a hormonal state receptive to supporting artificiallyintroduced rodent embryos. Such females are called pseudopregnant At 2.5dpc (days post coitum) up to 15 of the stem cell containing blastocystsare introduced into the uterine horn. For early stage embryos andmorula, such embryos are either cultured in vitro into blastocysts orimplanted into 0.5 dpc or 1.5 dpc pseudopregnant females according tothe embryo stage into the oviduct.

Chimeric pups developed from the non-human embryos including donor stemcells develop to term after the transfer, birth being dependent uponembryo age at implantation and species. The gametes of the live chimeranon-human animals are substantially or wholly derived from the donorstem cells.

The live chimera non-human animals are bred to generate offspring, andall offspring are heterozygous regarding the donor stem cell genome.Alternatively, the gametes of the chimera are collected and used for invitro fertilization (IVF) or artificial insemination (AI). The gametesisolated from the chimera can also be cryopreserved and stored usingmethods known in the art. Alternatively, the germ cells of the chimeraare collected, matured in vitro or in vivo and used for in vitrofertilization or artificial insemination.

In vitro fertilization (IVF) methodology is well-established. See, forexample, Nagy et al. 2002, Manipulating the Mouse Embryo: A LaboratoryManual, 3^(rd) edition, CSHL Press. IVF generally comprises collectingoocytes and sperm from a female and a male respectively, fertilizingoocytes from the female with sperm from the male and maintaining theresulting fertilized oocytes under suitable conditions for developmentof the fertilized oocytes into embryos. Embryos may be harvested atdifferent stages. The female may be superovulated before oocytes arecollected for IVF. Fertilization may be achieved by IVF,intracytoplasmic sperm injection or zona drilling. See, for example,Nagy et al. 2002; Byers et al. 2006, Theriogenology 65, 1716-26;Ostermeier et al. 2008, 3 (7) e2792. IVF can be a useful tool toincrease the numbers of embryos obtained from a single female.

Intracytoplasmic sperm injection (ICSI) may be used to improvefertilization rates or to achieve fertilization. The ICSI procedureinvolves removal of the cumulus cells surrounding oocytes and injectionof the sperm or haploid spermatids into the oocytes, ordinarily througha glass pipette (see Kimura and Yanagimachi 1995, Biol Reprod.53(4):855-62). Spermatids, spermatogonial stem cells and male germ cellscan be differentiated in vitro and then used for ICSI (Marh J et al.2003, Biol Reprod 69(1):169-76; Movahedin M et al. 2004, Andrologia36(5):269-76; Ogura A et al. 1996. J Assist Reprod Genet. 13(5):4-31-4;Shinohara T et al. 2002 Hum Reprod 17(12):3039-45; Chuma S et al. 2005Development 132(1):117-22).

As an alternative to collecting mature oocytes for IVF from a female,immature oocytes may be obtained and allowed to mature in vitro, atechnique known as “in vitro maturation”. As an alternative, follicles,e.g., primary follicle or germ cells, may be isolated from the femaleand cultured in vitro to obtain oocytes useful for fertilization. Inmammals, only a small fraction of immature oocytes develop into matureoocytes; the rest degenerate and die. By isolating immature oocytes fromanimals and allowing them to mature in vitro, one can obtain many moreoocytes suitable for IVF from a given female in a short time frame.Mammalian oocytes are known to undergo maturation in vitro. In the caseof mice, cattle and other mammals, in vitro matured oocytes have beenfertilized in vitro and given rise to normal healthy offspring whenembryos were transferred to an appropriate uterus (Schroeder and Eppig1984 Dev. Biol. 102:493; Sirard et al. 1988, Biol. Reprod. 39:546). Invitro maturation technique is well known in the art. See, for example,Chiu et al. 2003, Human Reprod. 18: 408-416 and O'Brien et al. 2003,Biol. Reprod. 68: 1682-1686.

Artificial insemination is a process of fertilizing, female animals bymanual injection or application of sperm. In such a procedure, maleanimals are not required at the time of insemination; stored spermobtained from the animals can be used (see Wolfe 1967 Lab Anim Care1967, 17(4):426-32 and Sato and Kumura, 2002, J Assist Reprod Genet.19(11):523-30).

Other methods that can be used to generate live offspring from thechimera including surgical oocyte retrieval, ovary transfer, ovarysplitting, ovary fragment transfer, in vitro maturation of oocytes,follicles, spermatogonial stem cells, in vitro differentiation of germcells, and in vitro differentiation of primordial cells.

Methods for making a chimeric non-human animal or chimeric non-humananimal embryo from a non-human animal donor stem cell and a hostnon-human animal embryo are provided which include introducing anon-human animal donor stem cell into a non-human animal host embryo,wherein the non-human animal host embryo includes (1) a TetR geneoperably linked to a ubiquitous promoter or a developmentally-regulatedpromoter that expresses the TetR gene in germ cells during an embryostage, and (ii) a Tet Operator (TO) operably linked to a ubiquitouspromoter or a developmentally-regulated promoter that expresses the TOgene in germ cells during an embryo stage and operably linked to anucleic acid encoding a deleter gene. At least one non-human animaldonor stem cell is introduced into the non-human animal host embryo, andthe non-human animal host embryo including the at least one donor stemcell is introduced into a pseudopregnant female non-human animal to begestated. During gestation tetracycline or a tetracycline derivative isadministered to the female non-human animal to induce expression of thedeleter gene in the non-human animal host embryo, resulting in ablationof non-human animal host embryo germ cells. The introduced donor cellsdifferentiate into gametes in the non-human animal host embryo,resulting in live born chimeric non-human animals wherein the germ cellsand/or gametes are solely or substantially derived from the donor stemcells.

Methods for making a chimeric mouse or mouse embryo from a mouse donorstem cell and a host mouse embryo are provided which include introducinga mouse donor stem cell into a mouse host embryo, wherein the mouse hostembryo includes (i) a TetR gene operably linked to a ubiquitous promoteror a developmentally-regulated promoter that expresses the TetR gene ingerm cells during an embryo stage, and (ii) a Tet Operator (TO) operablylinked to a ubiquitous promoter or a developmentally-regulated promoterthat expresses the TO gene in germ cells during an embryo stage andoperably linked to a nucleic acid encoding a deleter gene.

At least one mouse donor stem cell is introduced into the mouse hostembryo, and the mouse host embryo including at least one donor stem cellis introduced into a pseudopregnant female mouse to be gestated. Duringgestation tetracycline or a tetracycline derivative is administered tothe female mouse to induce expression of the deleter gene in the mousehost embryo, resulting in ablation of mouse host embryo germ cells. Theintroduced donor cells differentiate into germ cells and gametes in themouse host embryo, resulting in live born chimeric mice wherein the germcells and gametes are solely or substantially derived from the donorstem cells.

Methods for making a chimeric non-human animal or non-human animalembryo from a non-human animal donor stem cell and a host non-humananimal embryo are provided which include introducing a non-human animaldonor stem cell into a non-human animal host embryo, wherein thenon-human animal host embryo includes a nucleic acid encoding thymidinekinase operably linked to a developmentally-regulated promoter thatexpresses the thymidine kinase in germ cells during an embryo stage. Atleast one non-human animal donor stem cell is introduced into thenon-human animal host embryo, and the non-human animal host embryoincluding the at least one donor stem cell is introduced into apseudopregnant female non-human animal to be gestated. During gestationa thymidine analog, such as ganciclovir or a ganciclovir derivative, isadministered to the female non-human animal to ablate germ cellsexpressing HSV-tk or a truncated HSV-tk or Δ-TK in the embryos at anembryo stage from embryological stage E6 to E13, or a correspondingstage in a non-mouse species. According to embodiments, the pregnantfemale is treated with a thymidine analog to ablate germ cellsexpressing HSV-tk or a truncated HSV-tk or Δ-TK in the embryos at anyembryo stage from embryological stages E6.5 to E12.5 or a correspondingstage in a non-mouse species. The thymidine analog will be administered,once, or several times daily over several consecutive days, e.g. on daysE6.5, E7.5, E.8.5, E9.5, E10.5, E11.5 and E12.5; or intermittently, e.g.E6.5, E8.5, E10.5 or other intervals. For FIAU an amount from 10 to 50mg/kg/day will be administered. For GCV any amount from 20-100 mg/kg/daymay be administered.

Methods for making a chimeric mouse or mouse embryo from a mouse donorstem cell and a host mouse embryo are provided which include introducinga mouse donor stem cell into a mouse host embryo, wherein the mouse hostembryo includes a nucleic acid encoding thymidine kinase operably linkedto a developmentally-regulated promoter that expresses the thymidinekinase in germ cells during an embryo stage. At least one mouse donorstem cell is introduced into the mouse host embryo, and the mouse hostembryo including at least one donor stem cell is introduced into apseudopregnant female mouse to be gestated. During gestation a thymidineanalog, such as ganciclovir or a ganciclovir derivative, is administeredto the female mouse to ablate germ cells expressing HSV-tk or atruncated HSV-tk or Δ-TK in the embryos at any embryo stage fromembryological stages E6 to E13, or a corresponding stage in a non-mousespecies. According to embodiments, the pregnant female is treated with athymidine analog to ablate germ cells expressing HSV-tk or a truncatedHSV-tic or Δ-TK in the embryos at an embryo stage from embryologicalstage E6.5 to E12.5 or a corresponding stage in a non-mouse species. Thethymidine analog will be administered, once, or several times daily overseveral consecutive days, e.g. on days E6.5, E7.5, E.8.5, E9.5, E10.5,E11.5 and E12.5; or intermittently, e.g. E6.5, E8.5, E10.5 or otherintervals. For FIAU an amount from 10 to 50 mg/kg/day will beadministered. For GCV any amount from 20-100 mg/kg/day may beadministered.

Methods for making a chimeric mouse or mouse embryo from a mouse donorstem cell and a host mouse embryo are provided which include introducinga mouse donor stem cell into a mouse host embryo, wherein the mouse hostembryo is an F1 obtained by crossing a Deleter transgenic strain,containing a cytotoxic protein, such diphtheria gene, such asGt(ROSA)26Sor<tm1(DTA)Jpmb> with the Activator mouse strain containing asite-specific recombinase under the control of a developmentallyregulated promoter, such as FVB-Tg(Ddx4-cre)1Dcas. In all cellsexpressing the site-specific recombinase the cytotoxic protein will beactive and ablate such cells. One example for such a promoter is vasaand for the cytotoxic protein the diphtheria toxin fragment a (DTA).When examining the F1 offspring of the intercross ofGt(ROSA)26Sor<tm1(DTA)Jpmb> with FVB-Tg(Ddx4-cre)1Dcas, the resultingadult males are sterile and no sperm is detected in the vas deferentiaor the epididymides (see FIG. 7B). In FIG. 7A a testis section of a wildtype male mouse is shown with clear presence of spermatogonia (A),spermatocytes and round spermatids (B), elongating, spermatids (C) andSertoli cells (D). FIG. 7B shows a section of the testes from the F1intercross of Gt(ROSA)26Sor<tm1(DTA)Jpmb> with FVB-Tg(Ddx4-cre)1Dcaswhere only Sertoli cells (D) can be identified. To generate embryos,homozygous females of one strain (e.g. FVB-Tg(Ddx4-cre)1Dcas) aresuperovulated and mated with homozygous males of the other strain (e.g.Gt(ROSA)26Sor<tm1(DTA)Jpmb>). Alternatively, heterozygous mice may beused for one or both of the breeders. Embryos are collected 1.5 daysafter mating in the morning to isolate 2-cell stage embryos, or 2.5 daysafter mating in the morning to isolate 4-cell, 8-cell and 16-cell stageembryos, or 3.5 days after mating to isolate blastocysts by flushing theoviducts and uteri horns with embryo culture media, e.g. M2 media. Untilinjection the embryos are cultured under appropriate conditions. Forexample early stage embryos can be cultured in vitro to develop intolater stage embryos, such as morula and blastocysts. The embryos areinjected with stem cells as described above.

Chimera offspring are genotyped in the case when heterozygous parentswere used to create the host embryos to select only such mice whichcontain both DTA and Vasa promoter transgenes. These are used inconventional mating or as gamete donors for IVF. All sperm in malescontaining both DTA and Vasa promoter transgenes are derived from theintroduced stem cells. All oocytes in females from host embryos carryingboth, DTA and Vasa promoter transgenes, are derived from the stem cells.For example, R1 male ES cells derived from 129X1/SvJ and129S1/SV−1-+p+Tyr-cKit1S1−J/+mice (Nagy et al. 1993, PNAS USA 90,8424-8428), genetically engineered ES cells 129 S3/Svlmj+Tyr+c+Mfg EScells (7AC5/EYFP; SCRC-1033 ATCC), ES cells derived from BALB/c and BTBRT+tf/J have been injected into a VASA/DTA host embryo. In all cases, thesperm of male chimera have been derived from the injected ES cells.Further mouse iPS cells (clone 9.48B) were injected, producing chimericmice.

TABLE 12 Production of Chimeras using Cre-Vasa, DTA host embryos No. ofNo. of Fertile % Offspring Stem Cell Donor Stem Cell Chimeras ChimerasDerived R1 3 1 100% 7AC5/EYFP 2 1 100% BALB/cJ 3 1 100% BTBR T+ tf/J 6 4100% iPS 9.48B 2 Not tested Not tested

Standard analytical tools can be applied to test the identity of spermor offspring. Methods include but are not limited to sequencing, SNPanalysis, PCR technologies as well as protein markers, coat colormarkers, isozyme analysis (e.g. GPI, glucose phosphate isomerase iszoymeanalysis) and detection of any reporter genes or transgenes present inthe stem cells using standard methods well established in the art.

The donor stem cell may be a male stem cell (XY) or female stem cell(XX), or an XO stem cell. Male (XY), female (XX) and XO stem cells areintroduced into preimplantation host embryos using any of the hostsystems described above. In the case of male stem cells, the resultingmale chimera are advantageously used to produce offspring by breedingwith a suitable animal, including by artificial methods. In oneembodiment gametes of the male chimera are collected. In anotherembodiment germ cells or spermatogonial stem cells of the male chimeraare collected. In a further embodiment gametes, germ cells orspermatogonial stem cells are cryopreserved. In one embodiment, thefemale chimera is used to produce offspring by breeding. In anotherembodiment gametes of the female chimera are isolated. In one embodimentovaries of the female chimera are isolated. In another embodimentgametes or ovaries are cryopreserved.

In the case of XX and XO stem cells, the oocytes in the resulting femalechimera are derived from the donor stem cells and offspring are producedby breeding. In one embodiment gametes are isolated. In anotherembodiment ovaries are isolated. In a further embodiment gametes and/orovaries are cryopreserved. In another embodiment male chimera derivedwith XX or XO stem cells are used for breeding. In one embodiment germcells are isolated from such male chimera and used for breeding. Germcells may be matured by in vitro or in vivo techniques. In a furtherembodiment germ cells are cryopreserved.

Stem cells from one species of non-human mammal can be introduced to ahost of a different species of non-human mammal to produce germ cellsderived from the stem cells according to embodiments of the presentinvention. In one embodiment the stem cells are derived from rat,rabbit, marmoset, cattle, goat, sheep, pig, a rare and endangered mammalor an exotic mammal, such as zoological specimens. In one embodiment thestem cells are iPS cells.

An endangered mammal is a population of mammals which is at risk ofbecoming extinct because it is either few in numbers, or threatened bychanging environmental or predation parameters, such as elephants, largecats and non-human primates, including gray wolf, banded hare wallaby,jaguar Asian elephant, saiga antelope and northern white rhinoceros(Ceratotherium simum cottoni). Rare or endangered species” include butare not limited to any animal listed by any organization as beingthreatened or endangered, or any animal whose population, or habitat isthreatened, or any animal which is desirably breed in captivity. Forexample, lists of endangered species may be found at U.S. Fish andWildlife Service, Endangered Species Program or listed in the EndangeredSpecies Act (ESA).

Stem cells from one species of non-human mammal can be introduced to arodent host to produce germ cells derived from the stem cells accordingto embodiments of the present invention. In one embodiment the stemcells are derived from rat, rabbit, marmoset, cattle, goat, sheep, pig,an endangered mammal or an exotic mammal.

In one embodiment rat stem cells are introduced into a mouse host.Optionally, the rat stem cells are genetically modified.

In another embodiment marmoset stem cells are introduced into mousehost. The marmoset stem cells (Müller et al. 2009, Hum. Reprod 24 (6):1359-1372; Sasaki E et al 2005 Stem Cells 23:1304-1313) are optionallygenetically modified.

In one embodiment the stem cells are iPS cells, optionally geneticallymodified iPS cells.

Chimeric animals are produced in which the germ cells are derived fromthe stem cells. In a further embodiment the germ cells of the chimeraare isolated and matured in vitro or in vivo to generate gametes toproduce offspring.

In one embodiment rat stem cells are introduced into a mouse hostembryo, chimeras are generated and gametes are isolated from thechimeras and used for IVF or AI to produce offspring.

In another embodiment germ cells are isolated from a chimera andtransplanted into a donor rat depleted of its endogenous spermatogonialstem cells to allow the germ cells mature into sperm.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1 Stem Cell Chimera Production

Mouse ES or iPS cells are grown in media optimized for that particularline. Typically ES media contains 15% fetal bovine serum (FBS) orsynthetic or semi-synthetic equivalents, 2 mM glutamine, 1 mM Napyruvate, 0.1 mM non-essential amino acids, 50 U/ml penicillin andstreptomycin, 0.1 mM 2-mercaptoethanol and 1000 U/ml LIF (plus, for somecell lines chemical inhibitors of differentiation) in Dulbecco'sModified Eagle Media (DMEM). A detailed description of media is found inTremml et al., 2008, Current Protocols in Stem Cell Biology, Chapter1:Unit 1C.4. For review of inhibitors of ES cell differentiation, seeBuehr M., et al. 2003, Philosophical Transactions of the Royal SocietyB: Biological Sciences 358, 1397-1402.

For microinjection, ES or iPS cell are rendered to single cells using amixture of trypsin and EDTA, followed by resuspension in ES media.Groups of single cells are selected using a finely drawn-out glassneedle (20-25 micrometer inside diameter) and introduced through theembryo's zona pellucida and into the blastocysts cavity (blastocoel)using an inverted microscope fitted with micromanipulators.Alternatively to blastocyst injection, stem cells can be injected intoearly stage embryos (e.g. 2-cell, 4-cell, 8-cell, premorula or morula).Injection may be assisted with a laser or piezo pulses drilled openingof the zona pellucida. Approximately 9-10 ES cells (ES or iPS or epistem cells) are injected per blastocysts, or 8-cell stage embryo, 6-9 EScells per 4-cell stage embryo, and about 6 stem cells per 2-cell stageembryo. Following ES cell introduction, embryos are allowed to recoverfor a few hours at 37° C. in 5% CO₂, 5% O₂ in nitrogen or culturedovernight before transfer into pseudopregnant recipient females.Alternatively to ES cell injection, ES cells can be aggregated withmorula stage embryos. Such methods are well established and can be usedto produce stem cell chimeras. For a more detailed description seeManipulating the Mouse Embryo: A Laboratory Manual, 3^(rd) edition (A.Nagy et al. 2002, CSHL Press, ISBN-10: 0879695919, Nagy et al., 1990,Development 110, 815-821; U.S. Pat. No. 7,576,259, U.S. Pat. No.7,659,442, U.S. Pat. No. 7,294,754, Kraus et al. 2010, Genesis 48,394-399).

Pseudopregnant embryo recipients are prepared using methods known in theart. Briefly, fertile female mice between 6-8 weeks of age are matedwith vasectomized or sterile males to induce a hormonal state conductiveto supporting surgically introduced embryos. At 2.5 dpc (days postcoitum) up to 15 of the ES cell containing blastocysts are surgicallyintroduced into the uterine horn very near to the uterus-oviductjunction. For early stage embryos and morula, such embryos are eithercultured in vitro into blastocysts or implanted into 0.5 dpc or 1.5 dpcpseudopregnant females according to the embryo stage into the oviduct.Chimeric pups from the implanted embryos are born 16-20 days after thetransfer depending on the embryo age at implantation.

Example 2 In Vitro Fertilization (IVF) Method

IVF methodology is well-established. Briefly, to obtain oocytes suitablefemale oocyte donor mice are superovulated by injection of PMSG. After44-48 hr the animals are injected with hCG. Animals are euthanized at 13hrs post hCG injection. Cumulus enclosed oocytes within the oviductampulla are dissected out and collected using a beveled hypodermicneedle, and transferred into Fert (K-RVFE-50) COOKS Mouse In Vitro FertFertilization medium (MVF).

A suitable sperm donor male (typically 7-12 weeks of age) is euthanizedand the cauda epididymis with vas deferens is carefully removed avoidingcontaminating the sperm with blood. The sperm is released into 1 ml MVFmedia by making several cuts through the epididymides and vas deferentiausing a beveled hypodermic needle while holding the tissues with a pairof forceps and then allowing the sperm to flow out. Sperm count for agiven mouse is variable but a total of 25×10⁶ is average, of which 1×10⁴sperm/ml is adequate of IVF with 15-50 oocytes. For the IVF, sperm andoocytes are mixed in about 250 microliter of MVF medium.

After 4-6 hours incubation at 37° C. in 5% CO₂, 5% 0, in nitrogen thefertilized oocytes are collected and washed through two successive 150microliter media drops to remove cumulus cells and sperm. The fertilizedoocytes are cultured overnight at 37° C. in 5% CO₂, 5% 0, in nitrogen todevelop into 2-cell stage embryos. Embryos may be cultured longer, up todevelopment into the blastocyst stage. Pseudopregnant females at 0.5 dpcare used as the recipient for approximately 15 2-cell stageembryos/female. Pups from the introduced 2-cell stage embryos areexpected 18-21 days after the transfer as described for example in Byerset al. 2006, Theriogenology 65, 1716-26 and Ostermeier et al. 2008, 3(7) e2792.

Example 3 Administration of Tetracycline to Induce (Primordial) GermCell Ablation Allowing Preferential Stem Cell Colonization of theGermline

Example uses a strain containing transgenes having four elements; i) atissue specific promoter and enhancer promoter with a proximal enhancerand distal enhancer of mouse oct3/4, described in Ovitt and Schöler 1998Mol Hum Reprod. 4, 1021-31; ii) rtTA (reverse tetracycline-controlledtransactivator gene) a gene expressing protein which binds tetracyclineor its analogues (e.g. doxycycline) with poly A signal; and iii) theresponse element Tet-On (tetO; also called tetracycline-responsiveelement (TRE) or tet-operator or TO) which upon binding of rtTAcomplexes tetracycline and drives, iv) the DTA gene leading to celldeath. This strain of mouse is normally viable and fertile as thetransgene is inactive in the absence of tetracyclines. Such induciblesystems are well-established, for example as commercially available fromClontech and as described in Nishijima H et al. 2009 Biosci Trends(5):161-7.

To use this single strain as a host for stem cells providingpreferential colonization of the germline heterozygotes or homozygotesembryos are isolated and used as recipients for stem cells. To obtainhomozygote embryos the mouse strain is crossed using homozygote malesand females. To obtain a mixture of homozygote and heterozygote embryosone parent is homozygous and the other parent is heterozygous. To obtainheterozygote embryos the mouse strain is crossed either with a wild typemouse strain or any other genetically modified mouse strain. The femalesare superovulated to obtain a maximum number of preimplantation embryosand such embryos are used as stem cell recipient as described in Example1.

After the introduction of stem cells into the embryos (as outlined inExample 1) these are transferred into pseudopregnant females. Thepseudopregnant females are treated with doxycycline from embryonic dayE6.5 to E10.5 to induce the expression of DTA transgene in the embryos.Doxycycline is added in the drinking water at 5 mg/ml. Only in cellswhich are subject to doxycycline and are expressing oct3/4 (i.e. at≧E6.5 only primordial germ cells) will the DTA gene be activated causingcell death. This allows preferential colonization of stem cells to thegermline. After birth chimeric animals are analyzed regarding the stemcell contribution to the germline, which can be done by mating andgenetic analysis of the offspring. Alternatively, for males the spermcan be analyzed. Instead of mating, in vitro fertilization as describedin Example 2 or other artificial reproductive technologies can be used.

Example 4 Administration of Tetracycline to Induce (Primordial) GermCell Developmental Arrest Allowing Preferential Stem Cell Colonizationof the Germline

In this example a mouse strain mouse is used containing transgeneshaving three elements: i) TetR (reverse tetracycline-controlledtransactivator gene with poly A signal) is constitutively expressedunder the control of the CMV/chicken beta-actin (pCAG) promoter; ii) aTc operator (TO) sequence placed between the H1 promoter and, iii) ashRNA sequence of the target gene. In the absence of tetracycline thetranscription of shRNA from the H1 promoter is blocked by the binding ofthe TetR to the TO sequence. Upon providing tetracycline (doxycycline)the TetR binds with it and is unavailable to bind the TO sequencesinhibiting its binding of the TetR. This releases the H1 promoter andthe transcription of shRNA occurs causing a reduction of the cognategene. In this example tetracycline drives short hairpin RNA interference(shRNA) targeted to oct3/4 leading to its suppression in cellsexpressing oct/3/4 and arrest and/or death, i.e. host germ cells. shRNAwhich cause knockdown of oct3/4 are described for example in Velkey andO'Shea, 2003, Genesis 37, 18-24; Zafarana et al. 2009, Stem Cells 27,776-782 and by Clontech and Ambion, Life Technologies in RNA Resources.

Host embryos containing the transgenes TetR and oct3/4-shRNA can be usedas a host for stem cells allowing preferential colonization of thegermline. Preimplantation embryos are isolated for stem cell injection.After the introduction of stem cells in the preimplantation embryo (asdescribed in Example 1) and their subsequent transfer intopseudopregnant females the transgene present in the host embryos isinduced with doxycycline from embryonic day E6.5 to E10.5, e.g. byproviding doxycycline in the drinking water 5 mg/ml to recipient femalesat ≧E6.5 for 4 days. Upon tetracycline induction, all host embryoderived cells will express the shRNA transgene, however only thoseexpressing nominally oct3/4 (i.e. at ≧E6.5 primordial germ cells) willthe interfering shRNA cause a cell disruptive event leading todevelopmental cession and/or cell death. The period of doxycyclineinduction needs to be at least 4 days to cause total depletion of thehost embryos germ cell population, allowing preferential colonization ofstem cell derived germ cells. Induction is obtained by providingdoxycycline in the drinking water 5 mg/ml to recipient pregnant hostfemales at the times when the implanted embryos are at the ≧E6.5 stage.The treatment is given for 4 days.

After birth, male chimera offspring are selected for either mating,and/or sperm collection. For example, sperm can be cryopreserved beforefurther use. A sperm sample of the chimeric male mice can be genotypedto verify the stem cell contribution. Further, sperm can be used in invitro fertilization or artificial insemination to create embryos andlive offspring.

Example 5 Generation of Mouse Embryo Hosts by Site-SpecificRecombination Using Vasa Activator Strain

This approach uses two different mouse strains which when crossedproduce F1 offspring where endogenous germ cells are ablated early inthe development. Where ES or iPS cell have been introduced into the F1embryos derived from this cross, preferential expansion of ES or iPScell derived germ cells will occur followed by colonization of thegermline.

The Deleter strain is Gt(ROSA)26Sor^(tm1(DTA)Jpmb)/J (The JacksonLaboratory, stock 006331, abbreviated JR#006331) and contains a DTAtransgene with loxP sites, upon expression of Cre by driven by the Vasapromoter, a site specific recombination event occurs leading to the lossof EGFP expression, the expression of DTA and the death of any cell. TheDeleter transgenic strain JR#006331 was crossed with the Activator mousestrain FVB-Tg(Ddx4-cre)1Dcas/J (The Jackson Laboratory, stock 006954,abbreviated JR#006954). The Activator strain JR#006954 contains atransgene construct with the Vasa promoter directing Cre expression ingerm cells at least at embryonic stage E10.5. The Vasa promoter isdescribed in Gallardo et al., 2007, Genesis 45, 413-417. Upon crossinghomozygous JR#006954 with JR#006331 a recombination event occurs in alloffspring in cells where Vasa has been expressed (i.e. germ cells,Tanaka et al. 2000, Gene Dev 14, 841-853) leading to the activation ofDTA and the death of the germ cells in which it is expressed. Uponcrossing heterozygous JR#006954 (Vasa promoter transgene)×JR#006331(eGFP-DTA transgene) and examining male F1 offspring examiningcontaining both the eGFP-DTA and Vasa promoter transgenes no sperm arefound in the vas deferentia or the epididymides at >8 weeks of age.Frozen sections of the testis were prepared and examined, confirming theabsence of any spermatids, sperm or related cell types (see FIG. 7).

Homozygous JR#006954 females are superovulated and mated with JR#006331males. Embryos are collected 1.5 days after mating in the morning toisolate 2-cell stage embryos, or 2.5 days after mating in the morning toisolate 4-cell and 8-cell stage embryos, or 3.5 days after mating toisolate blastocysts by flushing the oviducts and uteri horns with M2media (Millipore # MR-015-D or SIGMA #M7167). Until injection theembryos are stored at 37° C., 5% CO₂ in KSOM medium (Millipore #MR-023-D). The embryos are injected with stem cells (see Example 1).

Alternatively, homozygous JR#006331 females are superovulated and matedwith JR#006954 males. Embryos are collected 1.5 days after mating in themorning to isolate 2-cell stage embryos, or 2.5 days after mating in themorning to isolate 4-cell and 8-cell stage embryos, or 3.5 days aftermating to isolate blastocysts by flushing the oviducts and uteri hornswith M2 media (Millipore # MR-015-D or SIGMA #M7167). Until injectionthe embryos are stored at 37° C., 5% CO₂ in KSOM medium (Millipore #MR-023-D). The embryos are injected with stem cells (see Example 1).

Alternatively, heterozygous mice may be used for one or both of thebreeders. Male offspring are genotyped to select only such mice whichcontain both eGFP-DTA and Vasa promoter transgenes. These are used inconventional mating or as sperm donors for IVF. All sperm in malescontaining both eGFP-DTA and Vasa promoter transgenes are derived fromthe introduced stem cells.

Example 6 Generation of Mouse Embryo Hosts by Site-SpecificRecombination Using Vasa Activator Strain Followed by Colonization ofthe Open Germ Cell Niche by introduced ES Cell

F1 blastocysts from (JR#003328×JR#006331) mating were produced by matingheterozygous JR#003328 with heterozygous JR#006331. Thus only 25% ofblastocysts are expected to carry the combination Vasa-Cre and DTA. Thegerm cells are eliminated only in blastocysts with both and DTA alleles.

ES cells were injected in all blastocysts isolated from theJR#003328×JR#006331intercross as described in Example 1. The mice WT,carrying only Vasa-Cre or only DTA were assayed as controls.

Approximately six 129 S3/Svlmj+Tyr+c+Mfg ES cells (7AC5/EYFP; SCRC-1033ATCC) were injected each into 14 blastocysts (129 53/Svlmj+Tyr+c+Mfg isa genetically modified subclone of R1 ES cells derived from(129X1×129S1)F1 mouse embryos.

Nine pups were born and all offspring were genotyped for both, eGFP-DTAand Vasa promoter transgenes. Two offspring, one male and one female,were positive for both transgenes. Vasa induced expression of Cre wouldoccur in germ cells at or before E10.5 days and onwards, leading to theactivation of by recombination the DTA gene and germ cell death. Adouble transgenic chimeric male was bred with DBA/2J females and 10litters were produced with a total of 87 offspring. All survivingoffspring were genotyped using SNP (single-nucleotide polymorphism) as agenetic fingerprint to determine the genetic origins of the animals(Petkov, P. M. et al. (2004) Genomics, 83 (5), 902-911). An example forthe SNP data is shown in Table 10 and SEQ ID NO:37 and SEQ ID NO:38. Inthe first two rows the sequence for both paternal strains is listed,i.e. the ES cell background129S1/SvImJ and for the females DBA/2J. Thethird row shows the data obtained from the offspring from mating thechimera with DBA/2J females. All offspring are heterozygous (het) wheredifferences exist between the parental strains. This approach was usedto genetically fingerprint 75 offspring from the chimera. All offspringwere determined to be paternally derived from the introduced geneticallymodified ES cell line R1 and maternally from the DBA2J oocytes; i.e. 129& DBA/2J F1, see Table 11.

TABLE 10 SNP example. Mouse Strain Sequence Data 129S1/SvImJ TCCCTG G AGAA T TGACC C G TATA T GGCCACGT DBA/2J TCCCTG T G GAA C TGACC T T TATA CGGCCACGT Offspring TCCCTG het het GAA het TGACC het het TATA hetGGCCACGT

TABLE 11 Genotype of offspring of Vasa-Cre, DTA double transgenic malechimera No. of tail Date of Confirmed ES Date of samples sample cellderived # Born Birth collected collection genotype 5 09 Mar. 2009 5 19Mar. 2009 5 8 01 Apr. 2009 7 08 Apr. 2009 7 6 09 Apr. 2009 6 23 Apr.2009 6 8 24 Apr. 2009 8 11 May 2009 8 7 01 Jul. 2009 7 24 Jul. 2009 7 1003 Jul. 2009 10 24 Jul. 2009 10 11 15 May 2009 11 27 May 2009 11 7 01Jun. 2009 7 01 Jul. 2009 7 9 26 Jun. 2009 6 24 Jul. 2009 6 16 07 May2009 16 11 May 2009 16

Example 7 Generation of Mouse Embryo Hosts by Site-SpecificRecombination Using Vasa Activator Strain Crossed with an Inverted loxPStrain

This approach uses two different fertile mouse strains which whenintercrossed produce F1 offspring where host germ cells are caused toundergo apoptosis upon Cre expression ˜E10.5 or before of embryonicdevelopment.

A deleter transgenic strain containing two loxP sites arranged ininverse orientation on chromosome 2; as described in Kmita et al., 2000,Nat Genet. 26:451-454 (The Jackson Laboratory, stock JR#012661,abbreviated JR#012661), is crossed with an activator mouse strain whichcontains a Vasa promoter region directing tissue specific appropriateexpression of Cre in germ cells at E10.5 or before of embryonicdevelopment (Tanaka, et al. 2000, Gene Dev 14, 841-853), available fromThe Jackson Laboratory, stock 006954 (abbreviated JR#006954). In the F1mice a recombination event occurs in all cells where Vasa has beenexpressed (i.e. germ cells) leading to a lethal chromosomalrecombination and cell death.

Homozygous JR#012661 females are superovulated and mated with JR#006954males. Embryos are collected 1.5 days after mating in the morning toisolate 2-cell stage embryos, or 2.5 days after mating in the morning toisolate 4-cell and 8-cell stage embryos, or 3.5 days after mating toisolate blastocysts by flushing the oviducts and uteri horns with M2media (e.g. Millipore # MR-015-D or SIGMA #M7167). Until injection theembryos are stored at 37° C., 5% CO₂ in KSOM medium (Millipore #MR-023-D). The embryos are injected with stem cells which are preparedas described in Example 1. For example, blastocysts are injected with aminimum of 5 stem cells, up to 25 stem cells. After injection, theblastocysts are cultured at 37° C. in 5% CO₂, 5% O₂ in nitrogen untiltransplantation. About 10 blastocysts per pseudopregnant female aretransferred into at 2.5 dpc the same day as the stem cell injections.Pups from the introduced blastocysts are expected about 18 days afterthe transfer. Male chimeras are bred and/or sperm are tested by PCR toascertain its stem cell origin.

Example 8 Generation of Rat Embryo Hosts with a Vasa-Cre Activator andDiphtheria Toxin Deleter Strain

Two transgenic rat strains are generated. One transgenic rat strain isinjected with a Vasa-Cre construct. As promoter, either the rat, mouseor human vasa promoter region are recombineered to the Cre recombinasegene with a functional polyA. The construct is injected into ratzygotes, and the offspring screened for presence and expression of thetransgene. Positive rats are tested for transmission of the functionaltransgene to the next generation and a homozygous rat strain isestablished. For the second transgene a vector is constructed such thatthe diphtheria toxin A fragment (DTA) gene is only active after the Crerecombinase has removed loxP flanked sequences. The loxP sites arelocated between the promoter and the DTA acne and prevent transcriptionof the DTA gene. The construct is injected into rat zygotes and theoffspring is screened for presence of the transgene. Positive rats aretested for transmission of the transgene to the next generation and ahomozygous rat strain is established.

To generate rat host embryos the two homozygous strains are intercrossedand preimplantation embryos are isolated. Such embryos are used tointroduce rat stem cells and to generate rat chimeras.

Example 9 Generation of Mouse Embryo Hosts by Site-SpecificRecombination Using a Vasa Activator Strain Followed by Colonization ofthe Open Germ Cell Niche by Introduced R1 ES Cells

F1 blastocysts were produced by mating homozygous ROSA26-DTA176 females(The Jackson Laboratory stock no. 010527; Wu et al. 2006, Development133:581-90) with homozygous B6.FVB-Tg(Ddx4-cre)1DCas/J (The JacksonLaboratory stock no. 012585) male mice. After intercrossing all derivedF1 embryos carry both, the Cre recombinase under a Vasa promoter controland the foxed DTA gene. This results in all F1 embryos eliminating theirown germ cells early in development. About 20 F1 blastocysts wereisolated from the JR#010527×JR#012585 and approximately six R1 stemcells (R1 ES cells, Nagy et al. 1993, PNAS USA 90, 8424-8428) wereinjected each F1 blastocyst. A total of 17 blastocysts were injected andtransferred to pseudopregnant females. Five pups were born, threefemales and two males. On low level chimeric male #2 and one mediumlevel chimeric male 3 were bred with 129S1/SvImJ (The Jackson Laboratorystock no. 002448) females. Chimera #2, which had very small testesproduced no offspring (FIG. 8). After examining testes and epididymis,no sperm were detected as shown in FIG. 9 A-C. Paraffin sections oftestes showing seminiferous tubules from infertile chimera #2 (FIG. 9A),compared with sperm productive and fertile chimera #3 (FIG. 9B),compared with control wild type testis (FIG. 9C). All seminiferoustubules from chimera #2 are empty and no sperm development isdetectable. In chimera #3 empty and filled seminiferous tubules aredetectable, while in wild type controls all seminiferous tubules arefilled.

As shown in FIG. 8, chimera #3's testes were smaller than control,however this animal was fertile producing six offspring. Offspring weretested for paternal origin by both coat color (agouti) and SNP(single-nucleotide polymorphism) genotyping. All offspring were shown tobe paternally derived from the R1 stem cells. To test more completelythe origin of the sperm and to confirm that an IVF approach can be usedto directly expand paternally derived offspring from chimeras, an IVFwas performed using chimera #3 as a sperm donor.

40 DBA/2J (The Jackson Laboratory stock no. 000671) females weresuperovulated, yielding 986 oocytes. These were used in an IVF withsperm isolated from chimera #3.

Sperm was examined by Hamilton Thorn NOS computerized semen analyzer(Hamilton Thorn, Beverly, Mass.), an Integrated Visual Optical Systemfor sperm analysis. For chimera #3 (date of birth Feb. 20, 2011) 15.8million sperm were in the sample, 4.4 million were motile (moving), 0.9million were progressively motile (travelling and not just moving).These data are consistent with that normally found in mice (see MousePhenome Database of The Jackson Laboratory).

The IVF using fresh sperm isolated from chimera #3 yielded 190 two cellembryos (19% of input oocytes), these were transferred into 15pseudopregnant females. Subsequently 75 pups were weaned. Using SNPanalysis (see Example 7 and Table 10) it was determined that alloffspring are paternally derived from the introduced ES cell line R1 andmaternally from the DBA2J oocytes; i.e. 129 & DBA/2J. F1.

Additionally, the testes of the chimeras #2 and #3 plus a control(JR#10527) animal were collected at 17 weeks of age and grosslycompared. FIG. 8 shows an image of a photomicrograph of the testes. Astriking size difference is apparent, with the sterile chimera #2 havingconsiderable smaller testis than fertile chimera #3 or wild typecontrol. For comparison a ruler in centimeter scale is included in FIG.8.

The testis of the chimeras #2 and #3 plus a wild type control mouse werecollected into Bouin's fixative, paraffin embedded, sectioned andstained with Periodic acid-Schiff (PAS) for histochemistry (see FIG.9A-C). A striking difference between the 3 groups is apparent. In thetestis of the sterile chimera #2 all seminiferous tubules are empty(FIG. 9A), i.e. no spermatogonial cells or sperm are present. In thetestis of the fertile chimera #3 many seminiferous tubules are showingactive sperm production, while also empty seminiferous tubules arepresent; i.e. partial colonization has occurred (FIG. 98). In contrastin the wild type control all seminiferous tubules are filled withdeveloping sperm cells (FIG. 9C).

Example 10 HSV-tk System to Generate Host Embryos

A transgenic rodent strain is constructed to express HSV-tk, a truncatedHSV-tk or Δ-TK under a developmentally regulated promoter, such as vasapromoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter,Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter, oct3/4promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiar promoteror TNAP promoter (see Table 1), which permits expression of HSV-tk ingerm cells, at least at some time during the embryonic stages from E6.5to E12.5, but not in other essential cell populations within this timeframe (expression prior and after this time is not relevant). Undernormal conditions expression of the HSV-tk gene has no significantharmful effects. Preimplantation embryos will be isolated from HSV-tktransgenic females and will be microinjected with donor stem cells. Toselectively ablate host germ cells in the developing embryo within thefemale the animals are treated with ganciclovir, FIAU or acyclovir,either orally or by injection (i.p. or i.v.), at a dose of 50 mg/kg/daywithin the gestation time from E6.5 to E12.5 in the case of mouse. Thetreatment time is at least for one day and may be continued for up to 7days. The drug treatment will ablate cells expressing HSV-tk, atruncated HSV-tk or Δ-TK and not affect other cells, including the donorstem cells. The resulting chimera will have all germ cells derived bythe donor stem cell.

Example 11 Generation of Mice from Female (XX) ES Cells Using the VasaHost Embryos

Blastocysts generated as described in Example 9 were injected withapproximately 16 female XX genetically modified ES cells, cell linecDK03 (Varlakhanova N V et al 2010, Differentiation, 80, 9-19). 68injected blastocysts were transferred into pseudopregnant females tomake chimeras as described in Example 10. Thirteen putative chimeraswere born (7 males and 5 females). At three weeks of age the femaleswill be superovulated and oocytes will be isolated and used in IVF. Theresulting offspring will be analyzed for ES cell contribution.Alternatively, female and male chimera will be used in breeding togenerate offspring.

Example 12 Generation of Mice from BTBR ES Cells Using the Vasa HostEmbryos

Blastocysts generated as described in Example 9 were injected withapproximately 16 male PB60.6 BTBR-derived stem cells. The ES cells wereisolated from the strain BTBR T+tf/J (The Jackson Laboratory stock no.002282), which is a model for autism Forty-one microinjected embryoswere transferred to pseudopregnant females as described in Example 10.12 pups were born (6 females and 5 males). These chimeras with variesdegrees of chimerism, from high (over 80% by coat color) to low (˜10% bycoat color) were mated with BTBR mice. To date offspring from 3 chimeraswere obtained. Thirty-nine pups have been obtained and all clearlyidentifiable by coat color that these are ES cell derived. The BTBR micehave a distinct coat color black and tan tufted, with a black back andtan belly. One of these fertile chimeras was a low percentage chimera asassessed by coat color (−10%).

Example 13 Introducing Rat ES Cells into a Mouse Host Embryo

Blastocysts generated as described in Example 9 are injected withapproximately six to ten male rat ES cells, line DAc8 (Li et al 2008,Cell, 135 (7) 1299-1310) obtained from MMRRC (University of Missouri).Resulting chimeras will be phenotypically examined at the age from 3-10weeks. Male chimeras will be sacrificed at the age of 7 weeks or olderand testes and epididymis will be isolated and examined for the presenceof germ cells and gametes. It is expected that rat germ cells andgametes are capable to develop in the mouse host environment. Femalechimeras will be sacrificed at 3 weeks of age or older and ovaries willbe isolated and examined for the presence of germ cells and gametes.Male chimeras will be sacrificed at 7-10 weeks of age and examined forsperm and colonization of the testis. Any present germ cells or gametesare expected to be derived from the rat ES cell. Germ cells and gametescan be used for breeding as described above.

Example 14 Introducing BALB/cByJ-PB150.18 ES Cells into the Vasa HostEmbryos

21 blastocysts generated as described in Example 9 were injected withapproximately six to ten male BALB/cByJ-PB150.18 ES cells. Ten pups wereborn. Three male chimeras were bred with BALB/cJ (JR#000651) females,but no offspring were produced. After examining the testes andepididymis, sperm were detected in one male. Sperm concentration andmotility was comparable to controls. An IVF with BALB/cByJ donor femalesfor oocytes using sperm from the male chimera yielded two cell embryosshowing that BALB/cByJ-PB150.18 is capable to colonize the germ layerand produce germ cells and gametes.

Example 15 Detection of the Vasa-Cre Transgene

DNA is isolated, either from the chimera or host embryo, oralternatively a DNA lysates may be used. About 10-300 ng genomic DNA areapplied to each PCR reaction. The Vasa-cre specific primers SEQ EDNO:40: CACGTGCAGCCGTTTAAGCCGCGT and SEQ ID NO:41 TTCCCATTCTAAACACCCTGAAand control primers as positive control to monitor that the PCR assayhas worked with SEQ ID NO:42: CTAGGCCAAGAATTGAAAGATCT and SEQ ID NO:43GTAGGTGGAAATTCTAGCATCATCC. The PCR is performed with an annealingtemperature of 67° C. and for 35 cycles. The PCR reaction is made withstandard PCR buffer, e.g. AB PCR buffer II, MgCl₂, dNTP, Taq DNApolymerase, 1 uM of the Vasa-Cre primers and 0.5 uM of the positivecontrol primers and the DNA to be tested. The PCR cycle regime is forexample, of one cycle 3 min at 94° C. followed by 35 cycles consistingof 30 s at 94° C., 1 min at 67° C. and 1 min at 72° C., finished by 2min at 72° C. The resulting product is separated by gel electrophoresison a 1.5% agarose gel. For Vasa-Cre, a 240 bp band is detectable and forthe positive control a 324 bp band. Alternatively quantitative PCR ormelt curve analysis or sequencing assays can be performed.

Example 16 Detection of the DTA Transgene

DNA is isolated, either from the chimera or host embryo, oralternatively a DNA lysates may be used. About 10-300 ng genomic DNA areapplied to each PCR reaction. Three primers are used, two detecting wildtype and one detecting Gt(ROSA)26Sor<tm1(DTA)> transgene. The primersare SEQ ID NO:44 GTTATCAGTAAGGGAGCTGCAGTGG; SEQ ID NO:45GGCGGATCACAAGCAATAATAACC and for transgene detection SEQ ID NO:46AAGACCGCGAAGAGTTTGTCCTC. The PCR is performed with an annealingtemperature of 64° C. and for 35 cycles. The PCR reaction is preparedwith KAPA PCR buffer, dNTP, KAPA Taq DNA polymerase (Kapa BiosystemsInc), 1 uM of each primer and the DNA to be tested. The PCR cycle regimeis for example, of one cycle 3 min at 94° C. followed by 35 cyclesconsisting of 30 s at 94° C., 30 s at 64° C. and 30 s at 72° C.,finished by 2 min at 72° C. The resulting product is separated by gelelectrophoresis on a 1.5% agarose gel. For the transgene, a 302 bp bandis detectable and for wild type a 415 bp band. For a heterozygous sampleboth bands, the 302 bp and 415 bp bands, are detectable. Alternativelyquantitative PCR or melt curve analysis or sequencing assays can beperformed.

Example 17 Development of Germ Cells or Gametes from Endangered Species

The example provided is pertinent to endangered mammals, including, butnot limited to, gray wolf, banded hare wallaby, jaguar, Asian elephant,saiga antelope and white rhinoceros.

The basic approach requires the creation of Induced Pluripotent Stemcells (iPS) which are a type of pluripotent stem cell artificiallyderived from a non-pluripotent cell, typically an adult somatic cells,e.g. skin cells, by inducing “forced” expression of specific genes. Theadult somatic cells here being derived from the endangered mammal andthe iPS cells being made using techniques well known in the art.

iPS cells are very similar to embryonic stem (ES) cells and have thecapacity to form viable chimeras, including the capability for germ celland gamete development. Here, iPS cells would be derived from both maleand female endangered mammals. Female derived iPS would be expected tocolonize efficiently the female germline, whilst male animal derived iPScells would efficiently colonize the male germline. The host embryo mayalso carry additional genes appropriate for the endangered mammal whichimprove the efficiently of germ cell and gamete development.

To achieve the development of germ cells and gametes from a endangeredmammal, iPS cells will be introduced into the morula and blastocyststage of F1 blastocysts produced by mating homozygous (JR#010527)females with homozygous (JR#012585) male mice, or other host embryos asdescribed above. In one combination all F1 embryos carry both, the Crerecombinase operably linked to a Vasa promoter and the foxed DTA gene.

Approximately six to sixteen male iPS derived cells will be injectedinto F1 blastocysts isolated from the JR#010527×JR#012585 intercross.Injected blastocysts will be transferred into pseudopregnant females tomake chimeras as described in Example 1.

At term, putative chimeras will be born. At ˜7 weeks of age animals canbe examined. Any germ cells or gametes in either the resulting male orfemale chimeras will be derived from the iPS cell and hence theendangered mammal. Upon maturation these germ cells or gametes will beused in IVF or ISCI, and the resulting embryos will be placed into asuitable host; i.e. one capable of carrying the resulting embryo toterm.

Example 18 Introducing Mouse iPS Cells into the Vasa Host Embryos

Forty-eight blastocysts generated as described in Example 9 wereinjected with approximately six to sixteen genetically modified mouseiPS cells (Varlakhanova et al 2010, Differentiation, 80 (1), 949).Twenty-two pups were born. To date two chimeras were detected andexperiments are ongoing to analyze germ cell contribution and fertility.

Sequences

SEQ ID NO: 1 Vasa Promoter - Mus musculusTGTGCCACCATGCCTGGCCCAGTTTCTTGCTTTGTATTATAAACTTTATAGCTGGTAAGACTCTGGACCCACACGTGTTTGATTTGTCTCTTCTCTCCTGAGACATTTTTTCCCAGTAACATTTCCTGAATTTTGTATTTTATTGTCTCTGCATCTGTGCTGAGACATAACATCTTCTGTTTCTTCTATTTGCCATGAACTGGGAGTTAAGTCTAAGAATTTGATTAAAGCTGAAGTTGAGTTTGAAATCAAACCTCTACATGTAGCGTTTTGTATTTCCCACTGTGATACATCAACCAGCACATGGTTCTTGGAGGTCTTTGCTTTGTGGTATAAAGTTGGCCCAGTGTGTTTAAATGTTGTCCTCTTTAAGGAGGTAGCTAGGTAAGATCACACGCCGGTTACCTACCTCCCTACTGATGCACTAACTGGTGTGTCTAGCCTAGCCTAAGGCCCTAGGGTACCATGCATCCCATAATAGCTTATGTGAGGCCCAACACATTTGTAGATAACAGCATCAGGTCATAGTGTCATAAGGTTGGACACCCCTGGCAGAGTTTCCTGTACCACACTATCTTCAAAAGAGCTAAAGAAGTAAGGTGGAAAAAAACAAACATCATAGTGTTCTGATTTTACTGTAACAGGAAAAGACTCACTGAAAAGATGCCAAGGCCCAGATTTCCCCTCCCACAATTCCAAGCTTCATGGAGAATGATACCTCCACTTTGGGGATGGAGAAGGAATTAAATCCTGACCACAGACTTGAAGCAGTAAGAACTTATCACCGTGATGTCAGTTCTGGGTGGAACCCTATGCATCCCGTGGCCCTGTACCTGCAGATGCTGCTGCTGGACCTGTGATAGATGGCCAGTACTACAGTTAGGAATCTTTCTGACAACTGCAGCTTTTAGAATGGAAATCAAAACTCCACCTACTGGATGGCACAACATGAAAGCAAGGACTGTGCCTTGCAGCTCAGGGCCAGCCTTAGTACTAGTCTCTGAGGGACCGTTGCTACCACCGGTTCCCAACCTGTAGCAGCACAACTATCCTGGACCAGGGTTCACGGATGGAGCTCAAGACCTTGACATCTGTCACAGTTATGCTCTGCACCATGAGGAAGTTGTCTCAGTTTGCCTTACTGCCACAGCACCGGCTGTGGGCTCAGCATTGTGACACTCAGAGCCACAGTTCTGGAGCAGGAACCAGGGCAGGTCCCTCTGGTGATTCTTTCTACAGTTCACGACCAGAGGCTGTTGGCTTTGGAAGGCACCTGGAGGTCTGTGCAAGCCCCGGACAGAGGCTTGTGTAGATGAAGGACCCTTTATAAAAAGCTCCCTAATTGAGTCTTGAAAAACTCACCOGCTGAGGATTTAGGAGAAACCTAAGCTTGGATCGCCTCCTAACACTGCCACAGGTAACCTGAATTTTGGTGCCATATAGTGTTAGAAACTATAGGCTGAGAGAGAGAGAGAAAAAAAAAACATTCTTTTTTCAATTTCTGAAAACAAACAAAACCAAAACAAGCCATATTATGAAGACCGGAATAAATACCTAATCCTTTGTCCTCGAATGACATCACACATCAATAAGAACCAAGGATGTTTAAGGAACTATGACCTCATCTGACAGTATAAATATAAAATGCCAGAAACCGTCAAGATAGTAACCAGTGTGGGTGGATGCTCATATGTGTACTGTATTAGTTACTTTTTCATTGTTGTGATAAAATACCACGACTAAAGCCTGACTTATAGGAGAAGGAATTTGTCCTGGCTTGTGTTTTCAGAGTGATATGACTCCATCATGACAGGGACGCATGGAAGCCAGCAACAGGTATGATGCTGGGGCAGCAAGCTGAGAGCTCACATCCGCCACTGAAGGCACAAAACAGCAAGTGTGAGCTGGAAGTGGTGTGAGGCTATATACTAGCAAAGGCTGCCCTACTGATACGCTGTCTCCAGGAGGACTGCATTCCCCAAGCTTCTCTGATCATCACAACCAACTGAGAACTAAATGGTCAAATACCTGGGTCAGTGGGGATCATTTCTCATTCAAACTTCCACAGATAATTAAAAGCAAATAAATGAACCAGGTTTAAGGAAACTTACTGAATTCCAATAGAAAATATAGAAATAAATCAATACTTTAGTAGATATAATTGACTGAGACATGGGGGCAAATTTTAAAAAGTTGAAATTCTTGATAATGAAGGTATACTAAACAAAATAAAATTGCATTAGGGAAGCATTGATGTTAGAGTAAATCAAGCAGAAGAGAGAATGAGTGAATTCCAACATAGGCCATTTGAAAATGCACAGAAGAGAAAAAGAATTAAGAGGAAGAAATGTACTCATGGTAACCTTGGAGCAACAGTGAAAGGGTAAGTAGGTTAGTGGGGATATGGACGGGACTTGAGAACACCAAAAGAGTTAATCGATGCAACTACGTTATGATCAAACACAGTCAGGTTCCAGGTTCTGCAGTAAACTGAATCTCCAGTTTTCACGTTATGTAGATCCATCTCCATGAATCTGGCCTGGCCTTGTGAATTTCTGGTAACTGATTAGTTGAAAGTTGCAAATATAATTGTTTAACTTCTTATGCTAGAATTCAAGAAGCCTTGCAACTATTACCCTGGTCCTTTGGAGCGATGTCCCTGACAAGTGTGTCCAGTTGAAATGCAGCATCCTTACAGAGTCTGACTGAGATTGTCCAACAACCTTCAGAGCCTAAGTCAGCCTCTTGGAAACTATTCAATTGTTCTGGCCATCCCAGCCACTAGATGCCAGAGTAGAGAAGTCACCTTGGATGTTAAACAAACTGGTATTGGAGGTTGGGGATAAACTCAGTAGTACAGAGCTTGCCTGGCACGCTCAAGTACCTAGGTTCAAACTCTGGCAATGCCAAAAAAGAAAAAAGTTGATAGCAAACTCTCAGATGATTCAAGTTCTAATTAGCATTTGAATGGAATTGTATGAGATGGTCTCCTGAGCCCTCTCAATCCCTGAGAGAGAATTTTTTTTGTAAGTCAATATATTATGCAATAGTTTGTTATATAGTTGTACTGGCTAATTTGGGTCAAGCTGGAGTTATCACAGAGAAAGGAGCTTCAGTTGGGGAAATGCCTCCATGAGATCCAACTGTAAGGCATTTTCTCAATTAGTGATCAAGGGGGAAAGGCCCCTTGTGGGTAGGACCATCTCTGGGCTGGTAGTCTTGGTTCTATAAGAGAGCAGGCTGAGCAAGCCAGTAAGGAACATCCCTCCATGGCCTCTGCATCAGCTCCTGCTCCTGACCTGCTTGAGTTCCAGTCCTGACTTCCTTTGGTGATGAACAGCAATGTGGAAAGTGTAAGCTGAATAAACCCTTTCCTCCCCAATTTGCTCAGTGGTCATGATGTTTTGTCCTGGAATAGAAACCTTGACTAAGACAATAGTTATAGGTAACAAGAAGAGAGCTGTGAACTCGTCATGTAGTTAAGTGTTTGCTAGATTTCCTATTGCATAGTTCCTATTTTCTCTTTATATTTGGAAAAAAATTGTGGTAACATACCTTAGACACTTATTGTTGTTCTCCTCCCTGACCTGCACTCAATGAATTCTCAGCCAAACTCTAGTGATGTCCTCCCCATTCAAAAGCTTCAACCTTCTAGTCTTTATTTTAAAAAATGTTATTATTACATTTATTTATTCTGTGGTGTGTGTGTGTGAGAGAGAGGATGGGAGCAGGTGTGTGTGTGTGTGTCGGTGTGTTTGTTAAATCAGAACATGAGTGTGTGTGTAAGACAGAGACGGGGTGAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCAGTGGATAAGTTGTAGAAGTCAGTTCTTTCCTTCCCTTACTCGGACCTCAGGGATCAAACTCAAGTTCTCAGGTTGCCAGGTACATGTCACCTGTOTTTGCTTTATTATTATTTAGTTCTGGCTTTTGAGCCTAAGTGTTATGTTACATAGGTTGACCTTGAATTAGCTATGCACTGAGGTCCTGATCCTCCTGCCTTTATCTTCCAAGTAATGGTTACAGGAATGGGCCACTGGACCTAGCAAGTGAACCTACCTGGTCACTCCAAAAATGCACAGCAAAGAATATACGTTTAAAAATAGGTTCATTTTAGGAAGTTTGTCCACATTTTAAATGACAGTTCTGTTAAAGTATACTGTGTTTTGTCCTTTGTTAAATGTGACTTTTAAAAGCAATTCACCTTAATAGCCTGGGCGACTACAGTGCTCACTGTATAAATGCTAGTGTGTTTTTGGTGCTGAAACAGAAAGGTGGCTCTAGAAAGCTGGAGTTCCTCATCTTTAAGTTCCAGACTGAGATATCTAGAACTTCTTCAAAAATCCAGGGAAAGGAAAGCGATGGAAGTGAAAATAAAAACAAGAACATGCTTTACATATATTTGATTGTGATCCCTTTGGCGGGTACTAGGAAAACCACGGATGGAATTTTCCTTCTTGAAAGAGGTGAGGAGCAGGCAGAATGTGAACATCTACTTAATGAGCTGAACTGGCCGGTGCCCTCAGAATTGTAAACAGGTTCACCACAAATCCAGGCCTTGGCAAACAGACCAAGTOTTCCTCTCTTCGGTTTTCTTTTTACAGACTGGCTTTCTTGACAACTTCAAGATGGAGTCTCATCCTTGCCCTTTTTATGGAGAGGAGAAGCATTGCTTCTAGTTGGTTTTAGTAGAGGAGTGAAGTGCATTTCTCAGATACAAAGAGAGCACTTGAGACGTTCAGACTCAGAATGGCCAAGCCTGGCACTTTGGGAGGTCAAGAGGAGGCTGGAACGGCTGGGAGAGAAAGCAATTAGATGTTCCACCCCTTTGGTTTTTCTCCAGACAGGGTTTCTCTGTGTAGCCCTGACTGTCCTGAAGTTTGCTCTGTAGACCAGGCTGGCCTCCACCCAGGGATTTGCCTGCCTCTGCCTCCCCGAGCCCCAGATTTTTATTTTTATTTATTTATATATCTATTTTAACTTTTGAATGAACACAATGGAATTGATGAGCCCTTGGAGAGAGAAACGGGATGTCGTGCGTGGCAGCCCCGGGGATCAGCTCACTCCCACAGGCCTCACAGGCCATGGAGCCAAGAGGCCTCCCTGCCTCGGCCTCGGCCTCGGCCTCAACAAAGGTGGAGAACGCGCAGGCCGTCCGTCCATGGGGCGGGAAGTCGCGCGCCGCGGCCGCTGATTGGCTGGCGGGCCCGGTCGCCTGATGCTATTTGTTGTCCCCGCGCCAATGACGCAGTCGGCGTCCCGGCGTCCGCCCGCACGTGCAGCCGTTTAAGCCGCGTCGGCCGGCCGCGAGGAGCCCGGGGAGCCTGGAGCGGAGASEQ ID NO: 2 c-kit Promoter - Mus musculusAGGGAGAGTGCTAGGAGGAAGAGGATCCAGGGTGAAGGGCCTGTGGGGGCTCCTGGTCTTAGAGGGCACAGCGCCCCCGGGATCAGCTTATTGCAGCCCGAGAGCCCCGGGCACTAGGCAGCGGGAGGGAGTGCGACCCGGGCGGGAGAAGGGAGGGGCGTGGCCACGAGCTGGGAGGAGGGCTGGAGGAGGGGCTGTCGCGCGCCGCTAGTGGCTCTGGGGGCTCGGCTTTGCCGCGCTCGGTGCACTTGGGCGAGAGCTGTAGCAGAGAGAGGAGCTCAGAGTCTAGCGCAGCCACCGCGATGAGAGGCGCTCGCGGCGCCTGGGATCTGCTCTGCGTCCTGTTGGTCCTGCTCCGTGGCCAGACAGGTGGGAAAGAGCGGCAGACAAGAGGACTGCACCCTCTGTGGGCGCAGCCCGGGTCCGGGSEQ ID NO: 3 Artificial sequence - Transcription factor binding sequenceGTTCTCACGTGGCCTGSEQ ID NO: 4 Artificial sequence - Transcription factor binding sequenceCTCACGTGGCSEQ ID NO: 5 Artificial sequence - Transcription factor binding sequenceAGGCAAGGCAACATAASEQ ID NO: 6 Artificial sequence - Transcription factor binding sequenceTTGTTCTCACGTGGCCTGTGSEQ ID NO: 7 Artificial sequence - Transcription factor binding sequenceGTGTTAACGTCTGAASEQ ID NO: 8 Artificial sequence - Transcription factor binding sequenceTTCCTGGTCAAGGTCAGASEQ ID NO: 9 Artificial sequence - Transcription factor binding sequenceCACAGCTGGGSEQ ID NO: 10 Artificial sequence - Transcription factor binding sequenceCTATAAACAGACCTCTSEQ ID NO: 11 Artificial sequence - Transcription factor binding sequenceGTCATAGATAAGCTTSEQ ID NO: 12 Artificial sequence - Transcription factor binding sequenceCACCGAGAAGTATGASEQ ID NO: 13 Artificial sequence - Transcription factor binding sequenceCAGCACTGCCTCATAGATGASEQ ID NO: 14 Artificial sequence - Transcription factor binding sequenceGGACCGCCATCTGCCGGGGASEQ ID NO: 15 Artificial sequence - Transcription factor binding sequenceGATCTGCCATCCTGCCTGCCSEQ ID NO: 16 Artificial sequence - Transcription factor binding sequenceCAATTCCTGGAACTCSEQ ID NO: 17 Artificial sequence - Transcription factor binding sequenceTTCCAGGAATTGCACCACCTGGTGSEQ ID NO: 18 Artificial sequence - Transcription factor binding sequenceTTCCCAGTAGTGGCGACCCCAAGASEQ ID NO: 19 Artificial sequence - Transcription factor binding sequenceCTGTTGTTCACCAGSEQ ID NO: 20 Artificial sequence - Transcription factor binding sequenceTGAGAGGGTTTCGGSEQ ID NO: 21 Artificial sequence - Transcription factor binding sequenceGGAAGTGGGTCACSEQ ID NO: 22 Artificial sequence - Transcription factor binding sequenceTGAGAGGGTTTCGGSEQ ID NO: 23 Artificial sequence - Transcription factor binding sequenceGAGTCCAGGTGTTGGGSEQ ID NO: 24 Artificial sequence - Transcription factor binding sequenceTCCACCAGGTGGTGCASEQ ID NO: 25 Artificial sequence - Transcription factor binding sequenceGACTGGGCAAAAGTTCASEQ ID NO: 26 Artificial sequence - Transcription factor binding sequenceTATTGTTTGTTTSEQ ID NO: 27 Artificial sequence - Transcription factor binding sequenceCAAGGCCTCTGGCGTTSEQ ID NO: 28 Artificial sequence - Transcription factor binding sequenceGTCGTCAATCATGCCSEQ ID NO: 29 Artificial sequence - Transcription factor binding sequenceCAAGCGTGTGSEQ ID NO: 30 Artificial sequence - Transcription factor binding sequenceTGGGGAACGTGTTCCCSEQ ID NO: 31 Artificial sequence - Transcription factor binding sequenceGTTAGCACGTGAAGGASEQ ID NO: 32 Artificial sequence - Transcription factor binding sequenceGGTGAGTCAGCSEQ ID NO: 33 Artificial sequence - Transcription factor binding sequenceAGCTGACTCACSEQ ID NO: 34 Artificial sequence - Transcription factor binding sequenceCTCATTTACATACSEQ ID NO: 35 Diphtheria toxin A fragment from Corynebacterium diphtheriaeCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATCSEQ ID NO: 36 Artificial sequence - RNAi/shRNA target sequence for mouse oct3/4GGATGTGGTTCGAGTATGGT SEQ ID NO: 37 Artificial sequence - SNP sequenceTCCCTGGAGAATTGACCCGTATATGGCCACGTSEQ ID NO: 38 Artificial sequence SNP sequenceTCCCTGTGGAACTGACCTTTATACGGCCACGTSEQ ID NO: 39 Oct3/4 Promoter - Mus musculusACTGTGAGGGGATGGAGCCTGGGTGCAGGTCTTATGGGGGTTGGGGGGTGGTTAGTGTCTAATCTACCAACCTGGACAACACAAGATGGAATACTGTGCTCTGAAAACGCAGAGCCAGCACTTCTCTGGGGTCTCTGGGGACATATCTGGTTGGGGCTCGGGGTCCCATGGTGTAGAGCCTCTAAACTCTGGAGGACTGGAGGTGCAATGGCTGTCTTGTCCTGGCCTTGGACATGGGCTGAAATACTGGGTTCACCCATATCTAGGACTCTAGACGGGTGGGTAAGCAAGAACTGAGGAGTGGCCCCAGAAATAATTGGCACACGAACATTCAATGGATGTTTTAGGCTCTCCAGAGGATGGCTGAGTGGGCTGTAAGGACAGGCCGAGAGGGTGCAGTGCCAACAGGCTTTGTGGTGCGATGGGGCATCCGAGCAACTGGTTTGTGAGGTGTCCGGTGACCCAAGGCAGGGGTGAGAGGACCTTGAAGGTTGAAAATGAAGGCCTCCTGGGGTCCCGTCCTAAGGGTTGTCCTGTCCAGACGTCCCCAACCTCCGTCTGGAAGACACAGGCAGATAGCGCTCGCCTCAGTTTCTCCCACCCCCACAGCTCTGCTCCTCCACCCACCCAGGGGGCGGGGCCAGAGGTCAAGGCTAGAGGGTGGGATTGGGGAGGGASEQ ID NO: 40 Artificial sequence - Primer sequence for genotypingCACGTGCAGCCGTTTAAGCCGCGTSEQ ID NO: 41 Artificial sequence - Primer sequence for genotypingTTCCCATTCTAAACACCCTGAASEQ ID NO: 42 Artificial sequence -Primer sequence for genotypingCTAGGCCACAGAATTGAAAGATCTSEQ ID NO: 43 Artificial sequence - Primer sequence for genotypingGTAGGTGGAAATTCTAGCATCATCCSEQ ID NO: 44 Artificial sequence - Primer sequence for genotypingGTTATCAGTAAGGGAGCTGCAGTGGSEQ ID NO: 45 Artificial sequence - Primer sequence for genotypingGGCGGATCACAAGCAATAATAACCSEQ ID NO: 46 Artificial sequence -Primer sequence for genotypingAAGACCGCGAAGAGTTTGTCCTCSEQ ID NO: 47 Diphtheria toxin A fragment (DTA) protein from CorynebacteriumdiphtheriaeMDPDDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRS LSEQ ID NO: 48 Diphtheria toxin A mutant, tox176 protein from CorynebacteriumdiphtheriaeEADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFDDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSEQ ID NO: 49 Herpes simplex virus thymidine kinase from Human herpesvirus 1ATGGCCAGCTACCCCTGTCACCAGCACGCCAGCGCCTTCGACCAGGCCGCTAGAAGCAGAGGCCACAGCAACAGAAGAACCGCCCTGAGACCCAGAAGACAGCAGGAGGCCACAGAGGTGAGACTGGAGCAGAAGATGCCCACCCTGCTGAGAGTGTACATCGATGGACCCCACGGCATGGGCAAGACCACAACAACCCAGCTGCTGGTGGCCCTGGGCAGCAGAGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCAGGTGCTGGGAGCCAGCGAGACCATCGCCAACATCTACACCACACAGCACAGACTGGACCAGGGCGAGATCAGCGCCGGCGACGCTGCCGTGGTGATGACCAGCGCCCAGATCACAATGGGCATGCCCTACGCCGTGACCGATGCCGTGCTGGCTCCCCACGTGGGCGGAGAGGCCGGCAGCAGCCACGCCCCTCCCCCTGCCCTGACCCTGATCTTCGACAGACACCCCATCGCCGCCCTGCTGTGCTACCCCGCCGCTAGATACCTGATGGGCAGCATGACACCCCAGGCCGTGCTGGCCTTCGTGGCCCTGATCCCCCCTACCCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACAGACACATCATGGCTTCGTACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGTTCTCGCGGCCATAGCAACCGACGTACGGCGTTGCGCCCTCGCCGGCAGCAAGAAGCCACGGAAGTCCGCCTGGAGCAGAAAATGCCCACGCTACTGCGGGTTTATATAGACGGTCCTCACGGGATGGGGAAAACCACCACCACGCAACTGCTGGTGGCCCTGGGTTCGCGCGACGATATCGTCTACGTACCCGAGCCGATGACTTACTGGCAGGTGCTGGGGGCTTCCGAGACAATCGCGAACATCTACACCACACAACACCGCCTCGACCAGGGTGAGATATCGGCCGGGGACGCGGCGGTGGTAATGACAAGCGCCCAGATAACAATGGGCATGCCTTATGCCGTGACCGACGCCGTTCTGGCTCCTCATATCGGGGGGGAGGCTGGGAGCTCACATGCCCCGCCCCCGGCCCTCACCCTCATCTTCGACCGCCATCCCATCGCCGCCCTCCTGTGCTACCCGGCCGCGCGATACCTTATGGGCAGCATGACCCCCCAGGCCGTGCTGGCGTTCGTGGCCCTCATCCCGCCGACCTTGCCCGGCACAAACATCGTGTTGGGGGCCCTTCCGGAGGACAGACACATCGACCGCCTGGCCAAACGCCAGCGCCCCGGCGAGCGGCTTGACCTGGCTATGCTGGCCGCGATTCGCCGCGTTTACGGGCTGCTTGCCAATACGGTGCGGTATCTGCAGGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGACGGCCGTGCCGCCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTATTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTACAACGTGTTTGCCTGGGCCTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCACGTCTTTATCCTGGATTACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGGGATGATCCAGACCCACGTCACCACCCCAGGCTCCATACCGACGATCTGCGACCTGGCGCGCACGTTTGCCCGGGAGATGGGGGAGGCTAACTAASEQ ID NO: 50 Herpes simplex virus thymidine kinase protein sequence fromHuman herpesvirus 1MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKMPTLLRVYIDGPHGMCKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A method of generating a non-human embryo or animal derived fromdonor stem cells, comprising: a) producing a preimplantation non-humanhost embryo incapable of developing endogenous gametes; b) introducingdonor stem cells into the preimplantation non-human host embryo; c)gestating the non-human host embryo of b) under conditions suitable fordevelopment of the embryo, thereby generating a chimeric non-humananimal having substantially all gametes and/or germ cells derived fromthe donor stem cells; and d) malting a non-human embryo or animal usingthe gametes and/or germ cells derived from the donor stem cells, therebygenerating a non-human animal derived from the donor stem cells.
 2. Themethod of claim 1, wherein the preimplantation non-human host embryoincapable of developing endogenous gametes is generated by a methodselected from the group consisting of: expressing a cytotoxic protein ingerm cells of the non-human host embryo to ablate endogenous germ cells;expressing an inhibitory RNA in germ cells of the non-human host embryoto ablate endogenous germ cells; and expressing a recombinase to exciseat least two inverted recombinase recognition sites placed in achromosome of a preimplantation non-human host embryo to ablate theendogenous germ cells.
 3. The method of claim 2, wherein thepreimplantation non-human host embryo comprises a transgene, thetransgene comprising a nucleic acid sequence encoding the cytotoxicprotein, the inhibitory RNA or the recombinase, the nucleic acidsequence encoding the cytotoxic protein, the inhibitory RNA or therecombinase operably linked to a developmentally regulated promoteractive in germ cells of the embryo during at least a portion of adevelopmental stage corresponding to embryonic day 6 to embryonic day 14in mice.
 4. The method of claim 3, wherein the non-human host embryo isa rodent embryo and the developmentally regulated promoter is active inrodent germ cells during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryo.5. The method of claim 2, wherein the non-human host embryo comprises atransgene, the transgene comprising a nucleic acid sequence encoding thecytotoxic protein operably linked to a developmentally regulatedpromoter or a ubiquitous promoter, the transgene further comprising atleast one cytotoxic protein inhibitory sequence operably linked to atleast two recombinase recognition sites; wherein the non-human hostembryo further comprises a second transgene comprising a nucleic acidsequence encoding a recombinase, the nucleic acid sequence encoding therecombinase operably linked to a promoter selected from the groupconsisting of: a developmentally regulated promoter and a ubiquitouspromoter, wherein at least either the first or second transgene isoperably linked to a developmentally regulated promoter.
 6. The methodof claim 2, wherein the non-human host embryo comprises a transgene, thetransgene comprising a nucleic acid sequence encoding the inhibitory RNAoperably linked to a developmentally regulated promoter or a ubiquitouspromoter, the transgene further comprising at least one inhibitory RNAinhibitory sequence operably linked to at least two recombinaserecognition sites; wherein the non-human host embryo further comprises asecond transgene comprising a nucleic acid sequence encoding arecombinase, the nucleic acid sequence encoding the recombinase operablylinked to a promoter selected from the group consisting of: adevelopmentally regulated promoter and a ubiquitous promoter, wherein atleast either the first or second transgene is operably linked to adevelopmentally regulated promoter.
 7. The method of claim 2, whereinthe preimplantation non-human host embryo comprises a transgene, thetransgene comprising a nucleic acid sequence encoding the cytotoxicprotein, the inhibitory RNA or the recombinase, the nucleic acidsequence operably linked to an inducible promoter.
 8. The method ofclaim 5, wherein the transgene is operably linked to a developmentallyregulated promoter selected from the group consisting of: vasa promoter,Dnd1 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter,GDF-3 promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter,Prdm1 promoter, Tex13 promoter, and Tiar promoter.
 9. The method ofclaim 6, wherein the transgene is operably linked to a developmentallyregulated promoter selected from the group consisting of: vasa promoter,c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter.
 10. The method of claim 3, wherein the transgene is operablylinked to a developmentally regulated promoter selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter.
 11. The method of claim 1,wherein the donor stem cells are derived from a first animal species andthe preimplantation non-human host embryo is a different second animalspecies.
 12. The method of claim 3, wherein the transgene comprises anucleic acid encoding Herpes simplex virus thymidine kinase operablylinked to a developmentally regulated promoter and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog to ablate the endogenous germcells.
 13. The method of claim 12 wherein the transgene is operablylinked to a developmentally regulated promoter selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter.
 14. The method of claim 2,wherein the cytotoxic protein is diphtheria toxin A fragment (DTA),attenuated DTA or tox-176.
 15. A method of generating a chimericnon-human animal, comprising: a) producing a preimplantation non-humanhost embryo incapable of developing endogenous gametes; b) introducingdonor stem cells into the preimplantation non-human host embryo; and c)gestating the preimplantation non-human host embryo of b) underconditions suitable for development of the embryo, thereby generating achimeric non-human animal having substantially all gametes and/or germcells derived from the donor stem cells.
 16. The method of claim 15,wherein the preimplantation non-human host embryo incapable ofdeveloping endogenous gametes is generated by a method selected from thegroup consisting of: expressing a cytotoxic protein in endogenous germcells of the preimplantation non-human host embryo to ablate theendogenous germ cells; expressing an inhibitory RNA in germ cells of thepreimplantation non-human host embryo to ablate the endogenous germcells; and expressing a recombinase to excise at least two inverteddisposed recombinase recognition sites in a chromosome in the embryo, toablate the endogenous germ cells.
 17. The method of claim 15, whereinthe preimplantation non-human host embryo comprises a transgene, thetransgene comprising a nucleic acid sequence encoding the cytotoxicprotein, the inhibitory RNA or the recombinase, the nucleic acidsequence encoding the cytotoxic protein, the inhibitory RNA or therecombinase operably linked to a developmentally regulated promoteractive in endogenous germ cells of the embryo during at least a portionof a developmental stage corresponding to embryonic day 6 to embryonicday 14 in mice.
 18. The method of claim 17, wherein the non-human hostembryo is a rodent embryo and the developmentally regulated promoter isactive in rodent germ cells during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.
 19. The method of claim 16, wherein the preimplantationnon-human host embryo comprises a transgene, the transgene comprising anucleic acid sequence encoding the cytotoxic protein operably linked toa developmentally regulated promoter or a ubiquitous promoter, thetransgene further comprising at least one cytotoxic protein inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further comprises a second transgenecomprising a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a promoterselected from the group consisting of: a developmentally regulatedpromoter and a ubiquitous promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter.
 20. The method of claim 16, wherein the non-human host embryocomprises a transgene, the transgene comprising a nucleic acid sequenceencoding the inhibitory RNA operably linked to a developmentallyregulated promoter or a ubiquitous promoter, the transgene furthercomprising at least one inhibitory RNA inhibitory sequence operablylinked to at least two recombinase recognition sites; wherein thenon-human host embryo further comprises a second transgene comprising anucleic acid sequence encoding a recombinase, the nucleic acid sequenceencoding the recombinase operably linked to a promoter selected from thegroup consisting of: a developmentally regulated promoter and aubiquitous promoter, wherein at least either the first or secondtransgene is operably linked to a developmentally regulated promoter.21. The method of claim 19, wherein the transgene is operably linked toa developmentally regulated promoter selected from the group consistingof: vasa promoter, Dnd1 promoter, Fkbp6 promoter, Fragilis promoter,Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanos2 promoter,Nanos3 promoter, Prdm1 promoter, Tex13 promoter, and Tiar promoter. 22.The method of claim 20, wherein the transgene is operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter.
 23. The method of claim 15, wherein thepreimplantation non-human host embryo comprises a transgene, thetransgene comprising a nucleic acid sequence encoding the cytotoxicprotein, the inhibitory RNA or the recombinase, the nucleic acidsequence operably linked to an inducible promoter.
 24. The method ofclaim 17, wherein the transgene is operably linked to a developmentallyregulated promoter selected from the group consisting of: vasa promoter,c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter.
 25. The method of claim 15, wherein the transgene comprises anucleic acid encoding Herpes simplex virus thymidine kinase operablylinked to a developmentally regulated promoter and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog to ablate the endogenous germcells.
 26. The method of claim 25 wherein the transgene is operablylinked to a developmentally regulated promoter selected from the groupconsisting of: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter.
 27. The method of claim 16,wherein the cytotoxic protein is diphtheria toxin A fragment, attenuatedDTA or tox-176.
 28. The method of claim 15, wherein the donor stem cellsare derived from a first animal species and the non-human host embryo isa different second animal species.
 29. A non-human host embryo,comprising: a transgene encoding a deletes gene, the transgeneconfigured to express a cytotoxic protein or inhibitory RNA inendogenous germ cells of the embryo.
 30. The non-human host embryo ofclaim 29, wherein the transgene comprises a nucleic acid sequenceencoding the cytotoxic protein or the inhibitory RNA, the nucleic acidsequence encoding the cytotoxic protein or the inhibitory RNA operablylinked to a developmentally regulated promoter active in germ cells ofthe embryo during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 in mice.
 31. Thenon-human host embryo of claim 29, wherein the non-human host embryo isa rodent embryo and the developmentally regulated promoter is active inrodent germ cells during at least a portion of a developmental stagecorresponding to embryonic day 6 to embryonic day 14 of a mouse embryo.32. The non-human host embryo of claim 30, wherein the transgenecomprises a nucleic acid sequence encoding the cytotoxic proteinoperably linked to a developmentally regulated promoter or a ubiquitouspromoter, the transgene further comprising at least one cytotoxicprotein inhibitory sequence operably linked to at least two recombinaserecognition sites; wherein the non-human host embryo further comprises asecond transgene comprising a nucleic acid sequence encoding arecombinase, the nucleic acid sequence encoding the recombinase operablylinked to a promoter selected from the group consisting of: adevelopmentally regulated promoter and a ubiquitous promoter, wherein atleast either the first or second transgene is operably linked to adevelopmentally regulated promoter.
 33. The non-human host embryo ofclaim 30, wherein the transgene comprises a nucleic acid sequenceencoding the inhibitory RNA operably linked to a developmentallyregulated promoter or a ubiquitous promoter, the transgene furthercomprising at least one inhibitory RNA inhibitory sequence operablylinked to at least two recombinase recognition sites; wherein thenon-human host embryo further comprises a second transgene comprising anucleic acid sequence encoding a recombinase, the nucleic acid sequenceencoding, the recombinase operably linked to a promoter selected fromthe group consisting of: a developmentally regulated promoter and aubiquitous promoter, wherein at least either the first or secondtransgene is operably linked to a developmentally regulated promoter.34. The non-human host embryo of claim 32, wherein the transgene isoperably linked to a developmentally regulated promoter selected fromthe group consisting of: vasa promoter, Dnd1 promoter, Fkbp6 promoter,Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011promoter, Nanos2 promoter, Nanos3 promoter, Prdm1 promoter, Tex13promoter, and Tiar promoter.
 35. The non-human host embryo of claim 33,wherein the transgene is operably linked to a developmentally regulatedpromoter selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter.
 36. The non-human host embryo of claim 29, wherein thetransgene comprises a nucleic acid sequence encoding the cytotoxicprotein or the inhibitory RNA and wherein the nucleic acid sequence isoperably linked to an inducible promoter.
 37. The non-human host embryoof claim 30, wherein the transgene is operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter.
 38. The non-human host embryo of claim 29,wherein the transgene comprises a nucleic acid encoding Herpes simplexvirus thymidine kinase operably linked to a developmentally regulatedpromoter and further comprising contacting endogenous germ cellsexpressing the Herpes simplex virus thymidine kinase with a thymidineanalog to ablate the endogenous germ cells.
 39. The non-human hostembryo of claim 38 wherein the transgene is operably linked to adevelopmentally regulated promoter selected from the group consistingof: vasa promoter, c-kit promoter, Dnd1 promoter, Dppa3 promoter, Fkbp6promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3 promoter,Mov1011 promoter, Nanog promoter, Nanos2 promoter, Nanos3 promoter,oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13 promoter, Tiarpromoter and TNAP promoter.
 40. The non-human host embryo of claim 29,wherein the cytotoxic protein is diphtheria toxin A fragment (DTA),attenuated DTA or tox-176.
 41. A method of producing a non-human hostembryo incapable of developing endogenous gametes, comprising:introducing a transgene encoding a cytotoxic protein or RNA interferencemolecule into the embryo; and expressing the transgene in endogenousgerm cells, thereby generating a non-human embryo lacking functionalendogenous germ cells.
 42. The method of claim 41, wherein the transgenecomprises a nucleic acid sequence encoding the cytotoxic protein or theinhibitory RNA, the nucleic acid sequence encoding the cytotoxic proteinor the inhibitory RNA operably linked to a developmentally regulatedpromoter active in germ cells of the embryo during at least a portion ofa developmental stage corresponding to embryonic day 6 to embryonic day14 in mice.
 43. The method of claim 41, wherein the non-human hostembryo is a rodent embryo and the developmentally regulated promoter isactive in rodent germ cells during at least a portion of a developmentalstage corresponding to embryonic day 6 to embryonic day 14 of a mouseembryo.
 44. The method of claim 41, wherein the transgene comprises anucleic acid sequence encoding the cytotoxic protein operably linked toa developmentally regulated promoter or a ubiquitous promoter, thetransgene further comprising at least one cytotoxic protein inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further comprises a second transgenecomprising a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a promoterselected from the group consisting of: a developmentally regulatedpromoter and a ubiquitous promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter.
 45. The method of claim 41, wherein the transgene comprises anucleic acid sequence encoding the inhibitory RNA operably linked to adevelopmentally regulated promoter or a ubiquitous promoter, thetransgene further comprising at least one inhibitory RNA inhibitorysequence operably linked to at least two recombinase recognition sites;wherein the non-human host embryo further comprises a second transgenecomprising a nucleic acid sequence encoding a recombinase, the nucleicacid sequence encoding the recombinase operably linked to a promoterselected from the group consisting of: a developmentally regulatedpromoter and a ubiquitous promoter, wherein at least either the first orsecond transgene is operably linked to a developmentally regulatedpromoter.
 46. The method of claim 41, wherein the transgene comprises anucleic acid sequence encoding the cytotoxic protein or the inhibitoryRNA and wherein the nucleic acid sequence is operably linked to aninducible promoter.
 47. The method of claim 42, wherein the transgene isoperably linked to a developmentally regulated promoter selected fromthe group consisting of: vasa promoter, c-kit promoter, Dnd1 promoter,Dppa3 promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter,GDF-3 promoter, Mov1011 promoter, Nanog promoter, Nanos2 promoter,Nanos3 promoter, oct3/4 promoter, Prdm1 promoter, Prdm14 promoter, Tex13promoter, Tiar promoter and TNAP promoter.
 48. The method of claim 44,wherein the transgene is operably linked to a developmentally regulatedpromoter selected from the group consisting of vasa promoter, Dnd1promoter, Fkbp6 promoter, Fragilis promoter, Fragilis-2 promoter, GDF-3promoter, Mov1011 promoter, Nanos2 promoter, Nanos3 promoter, Prdm1promoter, Tex13 promoter, and Tiar promoter.
 49. The method of claim 45,wherein the transgene is operably linked to a developmentally regulatedpromoter selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex13 promoter, Tiar promoter and TNAPpromoter.
 50. The method of claim 41, wherein the transgene comprises anucleic acid encoding Herpes simplex virus thymidine kinase operablylinked to a developmentally regulated promoter and further comprisingcontacting endogenous germ cells expressing the Herpes simplex virusthymidine kinase with a thymidine analog to ablate the endogenous germcells.
 51. The method of claim 50 wherein the developmentally regulatedpromoter is selected from the group consisting of: vasa promoter, c-kitpromoter, Dnd1 promoter, Dppa3 promoter, Fkbp6 promoter, Fragilispromoter, Fragilis-2 promoter, GDF-3 promoter, Mov1011 promoter, Nanogpromoter, Nanos2 promoter, Nanos3 promoter, oct3/4 promoter, Prdm1promoter, Prdm14 promoter, Tex, 13 promoter, Tiar promoter and TNAPpromoter.
 52. The method of claim 41, wherein the cytotoxic protein isdiphtheria toxin A fragment, attenuated DTA, or tox-176.
 53. A non-humanhost embryo incapable of developing endogenous gametes, the non-humanhost embryo comprising a transgene encoding a recombinase operablylinked to a developmentally regulated promoter active in germ cells anda transgene encoding diphtheria toxin operably linked to a ubiquitouspromoter, wherein the transgene encoding diphtheria toxin operablylinked to a ubiquitous promoter and being operably linked to at leasttwo recombinase recognition sites.
 54. A non-human host embryo incapableof developing endogenous gametes, the non-human host embryo comprising atransgene encoding a recombinase operably linked to a vasa promoter anda transgene encoding diphtheria toxin operably linked to a ubiquitouspromoter, wherein the transgene encoding diphtheria toxin is operablylinked to a ubiquitous promoter having a loxP-flanked stop cassetteoperably linked to at least two recombinase recognition sites.
 55. Amethod of producing a non-human host embryo incapable of developingendogenous gametes, comprising: breeding a first animal of a firstrodent strain comprising a transgene encoding a recombinase operablylinked to a developmentally regulated promoter and a second animal of asecond rodent strain carrying a transgene with recombinase recognitionsites operably linked to a nucleic acid sequence encoding a cytotoxicprotein or inhibitory RNA operably linked to a ubiquitous ordevelopmentally regulated promoter.
 56. The method of claim 55 whereinthe first rodent strain is a first mouse strain comprising a transgeneencoding Cre recombinase operably linked with a vasa promoter and thesecond rodent strain is a second mouse strain comprising a loxP-flankedstop cassette operatively linked with a transgene encoding diphtheriatoxin operably linked with a ubiquitous or developmentally regulatedpromoter.